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Labile ruthenium(ii) complexes with extended phenyl-substituted terpyridyl ligands: synthesis, aquation and anticancer evaluation.
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DOI: 10.1039/C5DT02446C
The present study demonstrated that the anticancer activities of labile Ru(II)
complexes can be efficiently tuned by chelating with different phenyl-substituted
Dalton Transactions Accepted Manuscript
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terpyridyl ligands.
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DOI: 10.1039/C5DT02446C
Cite this: DOI: 10.1039/c0xx00000x
PAPER
www.rsc.org/dalton
Huaiyi Huang, Pingyu Zhang, Yu Chen, Liangnian Ji* and Hui Chao*
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Ruthenium complexes have been considered as promising substitution of cisplatin in cancer
chemotherapy. However, the novel ruthenium-based therapies faced with some limitations, such as
unimpressive cytotoxicity toward solid tumor. Herein, we designed and synthesized phenyl-substituted
terpyridyl ruthenium(II) complexes [Ru(tpy)(bpy)Cl]+ (Ru1) [Ru(phtpy)(bpy)Cl]+ (Ru2) and
+
10 [Ru(biphtpy)(bpy)Cl] (Ru3) which exhibited sharply different anticancer activity. Ru1-Ru3 all
underwent moderate aquation in buffer solution and this process was significantly inhibited by high
chloride concentration. Ru3 was relatively hydrophobic and could be readily uptake by cancer cells as
quantified by inductively coupled plasma mass spectrometry (ICP-MS). Ru1 was non-cytotoxic (IC50
>100 µM) while Ru3 exhibited very promising cytotoxicity on both two-dimensional (2D) cancer cell
15 monolayer and 3D MCTSs. Antiproliferative assay revealed that Ru3 significantly inhibited cellular
DNA replication which induced ultimately apoptosis of cancer cells.
Introduction
DNA transcription and replication accelerates in cancer cells
which make cell nucleus an emerging target for chemotherapeutic
1-4
20 intervention.
Cisplatin, the widely used chemotherapy agent,
form Pt-DNA crosslink which distorting DNA structure, blocking
DNA replication and inducing cancer cells apoptosis.5 However,
severe side effects and drug resistance restrict its clinical
application and have fostered interest in exploiting alternative
6
25 metallodrugs.
During the last 20 years, ruthenium (II/III)
complexes have emerged in the literature and gradually regarded
as promising alternatives to cisplatin.7 So far, three Ru(III)
complex NAMI-A, KP1019 and KP1339 are undergoing clinical
trials.8 Ruthenium complexes have certain advantages over
30 platinum anticancer agents. Ruthenium complexes exhibit lower
ligand aquation propensity than platinum complexes. The
cationic ruthenium complexes provide better solubility while the
octahedral structures result in diverse DNA coordination modes,
including covalent binding, groove binding, electrostatic
9-10
35 interaction, intercalation and insertion.
Besides, ruthenium
complexes can mimic ferrum binding to transferrin and thus be
transported directly to cancer cells, since transferrin receptors
were over-expressed at the surface of cancer cells than normal
cells.11 Thus, ruthenium complexes and lower toxicity toward
40 normal tissue than cisplatin. Moreover, ruthenium complexes
induce cell death through mechanisms differ from cisplatin which
render them active against cisplatin-resistant cell lines.12-15
However, the novel Ru-based therapies also faced with some
limitations, such as poor water stability or unimpressive
16
45 cytotoxicity toward solid tumor.
Recently, cellular uptake efficiency of metal-complex has
This journal is © The Royal Society of Chemistry [year]
gradually gained more and more attention, since the limited
penetration of cytotoxic drugs into solid tumors leads to the
frequent failure of chemotherapy to completely eradicate tumors
17
50 in clinic.
Traditional 2D cancer cell monolayer is a simplified
and widespread cell model for in vitro anticancer drug screening.
However, cell monolayer inevitably presents limitations in
reproducing the complexity and pathophysiology of tumors.18-21
Cells within solid tumor reflect reduced drug penetration or
55 pathophysiological differences such as hypoxia region and
relatively slow cell cycling. As a result, some experimental drugs
may be exclusively effective in cancer cell monolayer but noneffective in solid tumors. For example, doxorubicin shows
limited penetration into the solid tumors with only 40-100 µm
22
60 diffusion from blood vessels.
Moreover, the discovery of
multidrug resistance (MDR) in solid tumor highlighted the
differences between cancer cell monolayer and solid tumor.23
Multicellular tumor spheroids (MCTSs) are 3D heterogeneous
cellular aggregates that have been gradually accepted as a valid
24-25
65 cell model to mimic the features of in vivo solid tumor.
MCTSs provide insight into metabolic properties similar to solid
tumor profiles such as nutrient and oxygen gradients,
hypoxic/necrotic regions, cell-cell matrix interactions and gene
expression.26-28 MCTSs thus bridge the gap between the
70 oversimplified cancer cell monolayer and the highly complex
nature of solid tumor. MCTSs optimize anticancer drug screening
and increase the accuracy to predict in vivo anticancer activity
before animal studies.
In this paper, we reported three terpyridyl Ru(II) complex with
+
75 different phenyl-substituted ligand: [Ru(tpy)(bpy)Cl]
(tpy =
2,2':6',2''-terpyridine,
bpy
=
2,2'-bipyridine,
Ru1),
[Ru(phtpy)(bpy)Cl]+ (phtpy = 4'-phenyl-2,2':6',2''-terpyri-dine,
[journal], [year], [vol], 00–00 | 1
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Labile ruthenium(II) complexes with extended phenyl-substituted
terpyridyl ligands: synthesis, aquation and anticancer evaluation†
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Ru2) and [Ru(biphtpy)(bpy)Cl]+ (biphtpy = 4'-biphenyl2,2':6',2''-terpyridine, Ru3) as anticancer agents. The aquation
reactivity, DNA binding activity, log Po/w values, cellular uptake
efficiency and cell cytotoxicity of Ru1-Ru3 were studied. We
5 found that the length of phenyl-substituent on terpyridyl ligand
had a dramatic effect on biological activity of terpyridyl Ru(II)
complexes.
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Experimental
Materials and Instrument
All solvents were of analytical grade. Ruthenium chloride
hydrate, 2,2′-bipyridine (bpy), 2,2':6',2''-terpyridine (tpy),
cisplatin, ethidium bromide (EB) and acridine orange (AO) were
obtained from Alfa Aesar. MTT and PBS were obtained from
Sigma-Aldrich. All other reagents and solvents were of high
15 purity and used as received. Stock solutions of cisplatin (3 mM)
were prepared in saline and Ru1-Ru3 (10 mM) were prepared in
DMSO. All stock solutions were stored at -20°C, thawed and
diluted with culture medium prior to each experiment.
Microanalysis (C, H, and N) was carried out with a Vario EL
1
20 elemental analyzer. H-NMR spectra were recorded on a Varian
INOVA Mercury-Plus 300 NMR spectrometer. Electrospray
mass spectra (ES-MS) were recorded on a LCQ system (Finnigan
MAT, USA). The spray voltage, tube lens offset, capillary
voltage and capillary temperature were set at 4.50 KV, 30.00 V,
o
25 23.00 V and 200 C, respectively, and the quoted m/z values are
for the major peaks in the isotope distribution. UV-Vis spectra
were
recorded
on
a
Perkin-Elmer
Lambda
850
spectrophotometer. Emission spectra were recorded on a PerkinElmer LS 55 spectrofluorophotometer at room temperature
10
30
Synthesis
The terpyridyl ligands phtpy and biphtpy were synthesized
according to the published literature29 by changing appropriate
substituted benzaldehyde.
Synthesis of Ru(N^N^N)Cl3 To 125 mL of ethanol in a 200
35 mL round-bottom flask was added 262 mg (1 mmol) of
RuCl3.3H2O and tpy (233 mg, 1mmol) phtpy (309 mg, 1mmol),
biphtpy(385 mg, 1mmol), respectively . The mixture was heated
at reflux for 8 h with vigorous magnetic stirring. After cooled to
room temperature, and the fine brown powder which had
40 appeared was filtered from the reddish yellow solution. The
product was washed with 3 × 30 mL of ethanol followed by 3 ×
30 mL of diethyl ether and air-dried. Yield: Ru(tpy)Cl3 61 %,
Ru(phtpy)Cl3 56%, Ru(biphtpy)Cl3 45 %, respectively. The crude
products were used without further purification.
45
Synthesis of [Ru(tpy)(bpy)Cl]ClO4 (Ru1) A mixture of
Ru(tpy)Cl3 (0.044 g, 0.10 mmol), bpy (0.015 g, 0.1 mmol),
triethylamine (1.0 mL), ethanol (9.0 mL) and distilled water (1.0
mL) was refluxed under argon for 10 h. After most of the ethanol
was removed by rotary evaporation, a brownish-red precipitate
50 was obtained by drop-wise addition of excess NaClO4 solution.
The product was purified by column chromatography on alumina
using acetonitrile-toluene (2:1, v/v) as eluent. Yield: 60.2%. ES＋
MS (CH3OH): m/z = 526.3 [M - ClO4] . Anal. calcd. for
C25H19Cl2N5O4Ru: C, 48.01; H, 3.06; N, 11.20. Found: C, 47.89;
1
55 H, 3.17; N, 11.04. H NMR (300 MHz, d6-DMSO): δ = 10.07 (d,
J = 5.7 Hz, 1H), 8.88 (d, J = 6.0 Hz, 1H), 8.78 (d, J = 8.1 Hz,
2 | Journal Name, [year], [vol], 00–00
2H), 8.66 (d, J = 8.1 Hz, 2H), 8.60 (d, J = 8.1 Hz, 1H), 8.33 (t, J
= 7.8 Hz, 1H), 8.19 (t, J = 7.8 Hz, 1H), 8.04 (t, J = 7.8 Hz, 1H),
7.96 (t, J = 7.8 Hz, 2H), 7.75 (t, J = 7.8 Hz, 1H), 7.60 (d, J = 5.4
60 Hz, 2H), 7.35 (t, J = 6.9 Hz, 2H), 7.30 (d, J = 5.4 Hz, 1H), 7.05
(t, J = 7.2 Hz, 1H).
Synthesis of [Ru(phtpy)(bpy)Cl]ClO4 (Ru2) A mixture of
Ru(phtpy)Cl3 (0.052 g, 0.10 mmol), bpy (0.015 g, 0.1 mmol),
triethylamine (1.0 mL), ethanol (9.0 mL) and distilled water (1.0
65 mL) was refluxed under argon for 12 h. After most of the ethanol
was removed by rotary evaporation, a brownish-red precipitate
was obtained by drop-wise addition of excess NaClO4 solution.
The product was purified by column chromatography on alumina
using acetonitrile-toluene (1:1, v/v) as eluent. Yield: 54.6%. ES＋
70 MS (CH3OH): m/z = 602.0 ([M – ClO4] ). Anal. Calcd for
C31H23Cl2N5O4Ru: C, 53.07; H, 3.30; N, 9.98%. Found: C, 52.96;
H, 3.45; N, 9.72%. 1H NMR (300 MHz, d6-DMSO) δ 10.08 (d, J
= 5.4 Hz, 1H), 9.16 (s, 2H), 8.92 (d, J = 8.2 Hz, 3H), 8.63 (d, J =
7.7 Hz, 1H), 8.34 (t, J = 6.9 Hz, 1H), 8.30 (d, J = 8.1 Hz, 2H),
75 8.06 (t, J = 7.5 Hz, 1H), 8.00 (t, J = 7.5 Hz, 2H), 7.77 (t, J = 5.7
Hz, 1H), 7.69 (d, J = 7.2 Hz, 2H), 7.61 (m, 3H), 7.38 (dd, J =
12.6, 6.3 Hz, 3H), 7.06 (t, J = 6.0 Hz, 1H).
Synthesis of [Ru(biphtpy)(bpy)Cl]ClO4 (Ru3) A mixture of
Ru(phphtpy)Cl3 (0.059 g, 0.10 mmol), bpy (0.015 g, 0.1 mmol),
80 triethylamine (1.0 mL), ethanol (9.0 mL) and distilled water (1.0
mL) was refluxed under argon for 24 h. After most of the ethanol
was removed by rotary evaporation, a brownish-red precipitate
was obtained by drop-wise addition of excess NaClO4 solution.
The product was purified by column chromatography on alumina
85 using acetonitrile-toluene (1:1, v/v) as eluent. Yield: 49.5%. ES＋
MS (CH3OH): m/z = 678.1 ([M – ClO4] ). Anal. Calcd for
C37H27Cl2N5O4Ru: C, 57.15; H, 3.50; N, 9.01%. Found: C, 57.29;
H, 3.66; N, 8.86%. 1H NMR (300 MHz, d6-DMSO) δ 10.09 (d, J
= 4.6 Hz, 1H), 9.23 (s, 2H), 8.89 – 8.97 (m, 3H), 8.63 (d, J = 8.7
90 Hz, 1H), 8.43 (d, J = 6.9 Hz, 2H), 8.35 (t, J = 9.1 Hz, 1H), 8.05 –
7.99 (m, 5H), 7.86 (d, J = 8.1 Hz, 2H), 7.78 (d, J = 7.2 Hz, 1H),
7.62 (d, J = 4.2 Hz, 2H), 7.54 (t, J = 7.6 Hz, 2H), 7.46 – 7.35 (m,
4H), 7.08 (d, J = 3.3 Hz, 1H).
Aquation assay
Aquation of Ru(II) complex was monitored by UV-vis
spectroscopy.30 Ru1-Ru3 were dissolved in DMSO and diluted
with PBS buffer to give 30 µM solutions. The absorbance was
recorded at 30 min intervals over 8 h at s310 K. Plots of the
change in absorbance with time were fitted to the appropriate
100 equation for pseudo first-order kinetics using Origin version 7.5
(Microcal Software Ltd.) to determine half-lives and rate
constants.
95
Gel electrophoresis assay
105
110
Gel electrophoresis experiments31: DL5000 DNA marker was
treated with different concentrations of Ru1-Ru3 in 10 mM
phosphate buffer (pH = 7.5), and the solution was kept at 37 °C
for 1 h. Then, 2 µL of a quench buffer solution was added. The
samples were analyzed by electrophoresis for 1.5 h at 80 V on a
0.8% agarose gel.
Log Po/w measurements
Log Po/w is the partition coefficient between octanol and water
determined by the flask-shaking method.32 An aliquot of a stock
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solution of Ru1-Ru3 in 100 mM aqueous NaCl (0.9% w/v to
restrain aqueous and saturated with octanol) was added to an
equal volume of octanol (saturated with 0.9% NaCl w/v)
respectively. The mixture was shaken overnight at 60 rpm at 298
5 K to allow partitioning. After the sample was centrifuged at 3000
rpm for 10 min, the aqueous layer was carefully separated from
the octanol layer for ruthenium analysis. The Ru concentration in
the aqueous phase was determined by ICP-MS and used to
calculate the [Ru]o/[Ru]w ratio.
10
Cell culture and cytotoxicity test
The human cervix carcinoma cell line HeLa, the hepatocellular
carcinoma cell lines Hep-G2 and BEL-7402, the lung carcinoma
cell lines A549 and A549/CDDP, and the human normal
hepatocyte cell line LO-2 were obtained from the Experimental
15 Animal Center of Sun Yat-Sen University (Guangzhou, China).
Cells were routinely maintained in DMEM (high glucose,
HyClone) supplemented with 10% FBS (fetal bovine serum,
HyClone), 50 U/mL streptomycin, and 50 ng/mL penicillin at 37
°C under a humidified atmosphere with 5% CO2/95% air.
20
For cell cytotoxicity assay, approximately 1×104 cells/well
were seeded in 96-well plates, followed by incubation in 5% CO2
at 37°C for 24 h. After addition of serially diluted solutions of
Ru1-Ru3, cells were than incubated for another 48 h. Finally,
cytotoxicity was evaluated by the MTT assay; 20 µL of MTT
25 solution (5 mg/mL in 1× PBS) was added to each well, followed
by incubation for 4 h. The optical density of each well was then
measured on a microplate spectrophotometer (Biorad, USA) at a
wavelength of 595 nm..
ICP-MS assay
HeLa cells were plated at a density of 5 × 106 cells per100 mm
Petri dish in 10 mL of culture medium. 33 On the second day, the
cells were exposed to 10 µM Ru1-Ru3. After 6 h of drug
exposure, the drug-containing medium was removed. The cells
were washed twice with PBS and trypsinized, counted and
35 divided into three portions. Half of the cells were centrifuged and
washed with PBS for cytoplasm and nucleus fractionation
analysis by a nucleus extraction kit (Pierce, Thermo) following
the manufacturer’s protocol. In the second portion, the cytoplasm
was extracted using a cytoplasm extraction kit (Pierce, Thermo).
40 The third half of the cells were used for Genomic DNA
extraction for detection on ruthenium-DNA adducts using the
Nucleon genomic DNA extraction kit (GE healthcare,
Amersham, U.K.). The samples were digested with 60% HNO3 at
RT for one day. Each sample was diluted with MilliQ H2O to
45 obtain 2% HNO3 sample solutions. The ruthenium concentration
in each part was determined by inductively coupled plasma mass
spectrometry inductively coupled plasma mass spectrometry
(Thermo Elemental, USA).
30
EdU assay
EdU labeling was performed by using the Click-it EdU Alexa
Fluor 594 imaging kit (Molecular Probes).33 After incubating
HeLa cells with 5 µM cisplatin and Ru1-Ru3 for 24 h, 10 µM
EdU (Molecular Probes) was added to the culture media and
incubated for another 24 h. HeLa cells in 96-well plates were
55 washed with 1× PBS, and 200 µL medium containing 10 µM
EdU was then added to each well. The cells were incubated
50
This journal is © The Royal Society of Chemistry [year]
overnight and then washed with 1× PBS, followed by cell
fixation with 4% polyphosphoric acid. After 15 min of
incubation, the cells were washed twice with 3% BSA, followed
60 by cell permeabilization using 0.5% Triton X-100 in 1× PBS and
incubation for 20 min. The cells were then washed twice with 3%
BSA, and 200 µL Click-iT reaction mixture was added, followed
by 30 min incubation and one wash with 3% BSA. For nucleus
staining, 200 µL of Hoechst 33342 solution was added to each
65 well, and cells were incubated for another 30 min and washed
twice with 200 µL of 1× PBS. The cells were finally imaged
under an inverted fluorescence microscope (Zeiss Axio Observer
Z1, Germany).
Acridine orange/ethidium bromide (AO/EB) staining
Cell apoptosis studies were performed with a staining method
using acridine orange (AO) and ethidium bromide (EB).34 HeLa
cells was incubated in the absence or presence of Ru1-Ru3 at a
concentration of 5 µM at 37 oC and 5% CO2 for 24 h. After 24 h,
cells were stained with AO/EB solution (100 µg/mL AO, 100
75 µg/mL EB).
Samples were observed under an inverted
fluorescence microscope (Zeiss Axio Observer D1).
70
Generation and analysis of MCTSs
MCTSs were cultured using the liquid overlay method as
reported by our lab previously.33 HeLa cells in the exponential
80 growth phase were dissociated by a trypsin/EDTA solution to
gain single-cell suspensions. A number of 2500 diluted HeLa
cells were transferred to 1% agarose-coated transparent 96-well
plates with 200 µL of DMEM containing 10% serum. The cells
would generate singlet MCTSs approximately 400 µm in
85 diameter at day 4 with 5% CO2 in air at 37 °C.
Cytotoxicity on 3D MCTSs
MCTSs of diameters approximate to 400 µm were treated with
ruthenium(II) complexes and cisplatin by carefully replacing
50% of the medium with drug-supplemented standard medium
35
90 using an 8-channel pipettor.
In parallel, for the untreated
MCTSs, we replaced 50% of medium of the solvent-containing
or solvent-free medium. Four MCTSs were treated per condition
and drug concentration and the DMSO volume was less than
0.5% (v/v). The MCTSs were then allowed to incubate for
95 another 72 h. The cytotoxicity of ruthenium complexes was
measured by ATP concentration with a CellTiter-Glo
Luminescent Cell Viability kit (Promega). After 20 minutes of
incubation, the MCTSs were carefully transferred into blacksided, flat-bottomed 96-well plates (Corning) and pipette mixed
100 for luminescence measurement on infinite M200 PRO equipment
(TECAN)
Live/dead viability/cytotoxicity assay
The live/dead assay of MCTSs was performed using the
LIVE/DEAD Viability/ Cytotoxicity Kit for mammalian cells
36
105 (Life Technologies).
Live cells were distinguished by the
presence of ubiquitous intracellular esterase activity, as
determined by the enzymatic conversion of the virtually nonfluorescent cell-permeant calcein AM to the intensely fluorescent
calcein (λex = 495 nm, λem = 515 nm). EthD-1 would enter cells
110 with damaged membranes and undergo a 40-fold enhancement of
fluorescence upon binding to nucleic acids, thereby producing a
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Scheme 1. Synthesis route and structures of Ru1–Ru3.
bright red fluorescence in dead cells (λex = 495 nm, λem = 635
nm). Thus it was possible to determine cell viability depends on
5 these physical and biochemical cell properties. After treatment
with Ru(II) complexes, MCTSs were incubated with calcein AM
(2 µM) and EthD-1 (4 µM) solutions for 30 min and imaged
directly using an inverted fluorescence microscope (Zeiss Axio
Observer D1, Germany).
10
Results and discussion
Synthesis and characterization
Ru1-Ru3 were synthesized by reacting the corresponding
precursor with one equivalent amount of 2,2'-bipyridine in
refluxing ethanol. All complex were purified by alumina column
15 chromatography and characterized by ES-MS, elemental analysis
and 1H NMR spectroscopy (Fig. S1-S6).
Aquation of Chloride Ligand
Aquation of Ru-Cl bond was the an activated step for labile
transition metal anticancer complex such as cisplatin which
20 provides the possibility for metal center to coordinate with
cellular macrobiological molecule such as DNA or protein. First
of all, the aquation process of Ru-Ru3 was tracked by UV-Vis
spectroscopy. As shown in Fig. 1A and Fig.S7-S8, Ru1-Ru3
gradually aquated in PBS buffer solution (PH=7.4). With
25 incubation time extended, there was an apparent hypochromicity
in the metal-to-ligand charge transfer (MLCT) band (440-600
nm), accompanied with a new absorbance band appeared (370440 nm). The time-dependent absorptions of Ru1-Ru3 at the
indicated wavelengths follow pseudo-first-order kinetics (insets
30 figure). The aquation rates of the complex follow the order:
Ru1 > Ru2 > Ru3 with a relative long half-life time (Ru1, t1/2 =
1.5 h, Ru2, t1/2 = 2.4 h, Ru3, t1/2 = 3.0 h). Moreover, the m/z peak
of [Ru-Cl+H2O]+ (Fig. 1B and Fig.S7-S8) also support the
equated Ru(II) complex after incubation.
35
It has been well illustrated that chloride concentration have a
significantly effect on the aquation of Cl-containing metal-based
complex. The aquation degree of chloride ligand in present of
different NaCl concentration was detected by 1H NMR (Fig. 2).
4 | Journal Name, [year], [vol], 00–00
40
Fig. 1 (A) Aquation of Ru3 in PBS buffer solution (pH = 7.4)
tracking by UV-vis spectra. (B) LC-MS spectra of the aquated
Ru3.
In the absent of NaCl, new peak in 1H NMR spectra appeared
with the incubation time extended and reached equilibrium. As
45 expected, aquation of Ru1-Ru3 were totally suppressed in
present of 100 mM NaCl solution, a concentration similar to
blood plasma (Fig. 2 and Fig. S9-S11). The aquation of Ru1 Ru3 in present of 4 mM NaCl (a concentration was similar to
nucleus) was more readily than in 22 mM NaCl (a concentration
50 was similar to cytoplasm). The dependence of aquation on
chloride concentration may allow them to be selectively activated
inside cancer cells with reduced side effects during transportation
to cancer cells. Finally, the pKa values of coordinated water were
also determinded. The pKa values have a significant influence on
55 its reactivity since Ru-OH bond are often much less labile than
Ru-OH2 bond. The pKa values of coordinated water were
determined to be 7.32 ± 0.02 (Ru1), 7.50 ± 0.03 (Ru2) and 7.65
± 0.03 (Ru3). Thus, Ru1-Ru3 were thermodynamically stable
and yet kinetically labile in buffer solution.
60
DNA binding studies
First of all, we determined whether Ru1-Ru3 would exhibit
stacking interaction in buffer solution at concentration between 1
to 100 µM. It was evident in Fig. S12-14 that no significant
stacking interaction was observed for Ru1-Ru3.
65
Gel electrophoresis could provide an intuitive view to
determine whether compounds exhibit DNA binding activity.37
Cisplatin bind DNA covalentlyy which result in reduced DNA
gel electrophoretic mobility.38 The interaction of plasmid DNA
with Ru1-Ru3 were investigated (Fig. S15). Ru3 was found to
70 impede the migration of pUC19 DNA with increasing complex
concentration. In contrast, Ru1 and Ru2 were less efficient
though both of them also formed covalently binding with DNA.
These results implied that Ru3 can bind DNA by intercalation
besides covalent binding.
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DOI: 10.1039/C5DT02446C
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DOI: 10.1039/C5DT02446C
Table 1 IC50 (µM) values of Ru1-Ru3.
25
BEL-
G2
7402
>100
>100
>100
LO2
Ru1
Ru2
Ru3
Cisplatin
HeLa
A549
A549R
HeLa
>100
>100
>100
>100
>100
69.2
45.3
79.2
>100
51.4
>100
10.2
5.6
7.4
3.7
4.1
3.3
8.5
9.6
11.8
12.4
6.4
45.2
10.5
50.7
MCTSs
water. By extending the phenyl-terpyridyl ligand, the log Po/w
value increased gradually. Ru3 (0.63) tend to be more
hydrophobic than Ru1 (-1.1) and Ru2 (-0.35). The positive log
Po/w value of Ru3 may facilitate its cell uptake efficiency and
enhance anticancer activities.
Cytotoxicity assay
MTT assay was used to evaluate the anticancer activity of Ru1Ru3. Five human tumor cell lines, HeLa, Hep-G2, BEL-7402
A549, A549R and a normal cell line LO-2, were determined
(Table 1). Ru1 was nontoxic toward all cancer cells tested as well
30 as normal cells (IC50 >100 µM). The introduction of phenyl in
tpy ligand resulted in slightly increase in anticancer activity with
IC50 values ranging from 40 - 80 µM. Surprisingly, with regard to
biphenyl-substituted Ru3, significant enhanced anticancer
activity was observed. The IC50 values of Ru3 toward cancer
35 cells were even lower than that of cisplatin. Ru3 was also active
against cisplatin-resistant A549R cells with IC50 value similar to
the A549 cells. Additionally, all Ru(II) complex exhibited lower
cytotoxicity toward the normal cell line than cisplatin.
Cell Uptake Analysis
The high sensitivity of ICP-MS (inductively coupled plasma
mass spectrometry) makes it a practical method to detect and
identify metal-based complex within cells. The accumulation of
Ru1-Ru3 into nucleus and cytoplasm were studied. The total
ruthenium concentration within cells was in excellent agreement
45 with log Po/w data in the order Ru3 > Ru2 > Ru1. For all
complexes, the highest concentration of ruthenium was found in
the cytoplasm (Fig. 3). As the length of terpyridyl ligand
extended, the amount of ruthenium accumulated in nucleus
increased significantly. Since Ru1-Ru3 underwent aquation and
50 bound DNA covalently, genome DNA of HeLa cells after drug
treatment was isolated to determine the levels of DNA-bound
ruthenium within cells. Although the concentration of DNAbound ruthenium was not very high even for Ru3, the correlation
40
Fig. 2 Aquation of Ru1-Ru3 (A, B and C, respectively) in
present of different concentration of NaCl detected by 1H NMR.
In recent years, MALDI-TOF MS (martix-assisted laser
desorption ionization-time of flight mass spectrum) has been used
to determinded the formation of metal-DNA adducts.39 The
covalent binding of Ru1-Ru3 in present of double-stranded
oligonucleotides OD1 and OD2 were investigated. As shown in
Fig. S16 free oligonucleotides OD1 ([M-H]-) and OD2 ([M-H]-)
10 could
be clearly oberved. The spectra of Ru3-bound
oligonucleotides increased gradually whereas the peak of OD1
and OD2 decreased as incubation time extended. No peak at m/z
values over 6000 could be observed, suggesting that none of the
Ru(II) complexes formed interstrand cross-linking adducts. The
15 reduced peak intensity of oligonucleotides after adding Ru(II)
complexes confirmed covalently binding activity of Ru1-Ru3
with DNA.
5
Hydrophobicity Measurements
Log Po/w value is the partition coefficient between octanol and
This journal is © The Royal Society of Chemistry [year]
55
Fig. 3 Cellular ruthenium distribuction within HeLa cells..
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20
Hep-
Complexes
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Fig. 4 EdU antiproliferation assay. (A) cells without treatment,
(B) cells treated with 10 µM cisplatin, (C−E) cells treated with
Ru1-Ru3 (5 µM). Blue fluorescence represented Hochest 33342.
5 Red fluorescence represented EdU. Scale bars = 100 µm.
Fig. 5 HeLa cells were stained by AO/EB and observed under a
fluorescence microscope after 48 h drug exposures. (A) cells
without treatment, (B) cells treated with 10 µM cisplatin, (C−E)
45 HeLa cells treated with Ru1-Ru3 (5 µM). Scale bars = 100 µm.
between nucleus accumulation and cell cytotoxicity was apparent.
Ru3 with the most extended terpyridyl ligand exhibited the
highest level of ruthenium within nucleus and most cytotoxic.
metabolic and proliferative gradients found in solid tumors, such
as the altered responsiveness of chronically hypoxic tumor cells,
and multidrug resistance to chemotherapy drugs.44-45 Several
methods have been designed to generate tumor spheroids, among
50 which the agarose-coated liquid-overlay 96-well plate culture
provided an easy-handling and high throughput protocol for
generating single MCTSs in each 96 well.46-48 This method
exhibited desirable characteristics: (i) 96-well suspension culture;
(ii) a single spheroid per well; (iii) high reproducibility; and (iii)
55 simple harvesting for further analysis.
Antiproliferative Effect on HeLa Cells
EdU (5-ethynyl-2′-deoxyuridine) was a thymidine analog that can
incorporat into DNA replication, serving as a fluorescence
marker of active proliferating cells.41-43 EdU detection relies on a
simple and quick click reaction that does not necessitate a DNA
denaturation step like BdrU detection. For cells without drug
15 treatment, a large number of newly replicated DNA were
detected in the cell nuclei (Fig. 4), while DNA replication process
was dramatically reduced after incubating with cisplatin. For
Ru(II) complex, only Ru3 could distinctly inhibit DNA synthesis
process, while Ru1 and Ru2 were less effective. This result was
20 in agreement with cell uptake assay since only Ru3 can penetrate
into cell nucleus.
Then, we identified the cellular response after treatment with
the Ru(II) complex using the acridine orange/ethidium bromide
(AO/EB) dual staining assay (Fig. 5). AO is a vital dye and can
25 stain both live and dead cells. EB stains only cells that have lost
their membrane integrity. Under the fluorescence microscope,
live cells appear green. Necrotic cells stain orange red but have a
nuclear morphology resembling that of viable cells. Apoptotic
cells appear green, and morphological changes such as cell
30 blebbing and formation of apoptotic bodies will be observed.
After a 48 h treatment, Ru1 and Ru2 (5.0 µM) did not induce
apparent cell apoptosis because the cells were only stained by AO
(green). A large amount of apoptosis cells appeared after
incubation with Ru3 (5.0 µM) or cisplatin (10.0 µM).
10
35
Generation and analysis of MCTSs
In recent years, 3D multicellular tumor spheroids (MCTSs)
functioned as a better choice for in vitro negative anticancer drug
selection rather traditional cancer cell monolayer. MCTSs
restores the in vivo-like extracellular matrix (ECM) and is
40 capable of mimicking therapeutic problems associated with
6 | Journal Name, [year], [vol], 00–00
Cytotoxicity on 3D MCTSs cancer model
Cancer cells within solid tumor are generally less sensitive to
chemotherapeutics than cultured cancer cell monolayer. MCTSs
with diameters of approximately 400 µm have been suggested to
60 be a better choice than small MCTSs because they can better
resemble the pathophysiological conditions of solid tumors, such
as the specific hypoxic areas in the center and proliferation
gradients than small spheroids.49 We thus tested the cytotoxicity
of Ru-Ru3 and cisplatin on MCTSs with diameter around 400
65 µm. The IC50 values of cisplatin (50 µM) were almost 5 times
higher than treating with cancer cell monolayer (10.5 µM),
indicated significant multicellular drug resistance. Under the
same conditions, the IC50 values of Ru3 was 8.5 µM, nearly 3
times higher than the concentrations used with the cancer cell
70 monolayer. In contrast, Ru1 and Ru2 were totally inactive
against MCTSs (above 100 µM). These data indicated that Ru3
exhibited superior anticancer activity than cisplatin no matter on
2D cancer cell monolayer or 3D MCTSs.
The survival condition of MCTSs following drug treatment
75 was investigated by live/dead viability/cytotoxicity assay after
exposure to drugs. The virtually non-fluorescent cell-permeant
calcein AM will change to a green fluorescent species by
intracellular esterases in living cells. In contrast, EthD-1 could
simply enter dead cells and emit red fluorescence upon binding to
50
80 nucleic acids.
As shown in Fig. 6, untreated MCTSs as well
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DOI: 10.1039/C5DT02446C
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DOI: 10.1039/C5DT02446C
effective method to improve hydrophobicity of Ru(II) complexes
and thus enhance cell uptake efficiency and anticancer activity.
Conclusions
In conclusion, this study presented a valid procedure to increase
anticancer activity of terpyridyl Ru(II) complex. Ru1-Ru3 shared
different length of phenyl-substituted tpy ligand exhibited a huge
different in anticancer activity. Ru3 with biphenyl-tpy ligand
45 exhibit the higher DNA binding affinity and anticancer activity
than Ru1 and Ru2. The positive log Po/w value, high cell uptake
efficiency and nucleus distribution of Ru3 may contribute to its
excellent anticancer activity on cell monolayer and 3D MCTSs.
Cell proliferation assay and cell apoptosis experiments revealed
50 that Ru3 mainly induced cancer cells apoptosis. Taken together,
our research opened new approach for the design of terpyridyl
Ru(II) anticancer complex.
Acknowledgments
This work was supported by the 973 program (No.
2015CB856301), the National Science Foundation of China
(Nos. 21172273, 21171177, 21471164, and J1103305), the
Program for Changjiang Scholars and Innovative Research Team
in the University of China (No. IRT1298), and the National High
Technology Research and Development Program of China (863
60 Program, 2012AA020305).
55
Fig. 6 Calcein AM and EthD-1 dual-staining on drug-treated
HeLa MCTSs. (A) MTCSs without treatment, (B) MTCSs treated
with 10 µM cisplatin, (C) MTCSs treated with 50 µM cisplatin,
5 (D−F) MTCSs treated with Ru1-Ru3 (10 µM), respectively.
Scale bars = 200 µm.
as MCTSs treated with 10 µM cisplatin emitted steady green
fluorescence from the whole spheroid, suggesting no cell death
10 occurred within MCTSs. The dead cells appeared after treated
with 50 µM cisplatin. The green fluorescence significantly
weakened and bright red fluorescence appeared when the MCTSs
were treated with 10 µM of Ru3. Besides, the volume of MCTSs
reduced significantly compared with untreated MCTSs,
15 indicating severe cell
damage had occurred. In contrast,
complexes Ru1 and Ru2 (100 µM) did not show any impressive
cytotoxic effect on the MCTSs.
Structure-activity relationship
Compared with organic small molecules, metal complexes offer
several distinct advantages as therapeutic agents or biomolecular
probes.51-53 Our group have previously reported coordinatively
saturated polypyridyl Ru(II) complexes for anticancer drug
screening. Although these Ru(II) complexes exhibited excellent
in vitro DNA transcription inhibition or topoisomerase inhibitory
25 activity, most of which presented impressive cytotoxicity when
evaluated against cancer cells (IC50 between 50-490 µM).54-58
Such unexpected results impeled us to explore the intrinsic
structure-anticancer activity relationship of polypyridyl Ru(II)
complexes with the hope to increase anticancer activity. Barton et
30 al systematically explored the cellular uptake and localization of
a series of Ru(II) complexes containing intercalative ligand, and
found that the complex with the greatest lipophilicity exhibited
the greatest uptake.32 However, while changes in hydrophobicity
can modulate cellular uptake, this chemical property can also lead
35 to cytoplasm localization of the complex and reduced nuclear
targeting. In the present study, the above results indicate that
extending the tpy ligand with phenyl-substituent may be an
20
This journal is © The Royal Society of Chemistry [year]
Notes and references
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of
Chemistry and Chemical Engineering, Sun Yat–Sen University,
Guangzhou, 510275, P. R. China. Email: cesjln@mail.sysu.edu.cn,
65 ceschh@mail.sysu.edu.cn
† Electronic Supplementary Information (ESI) available: Figures of ESMS, 1H-NMR, DNA gel electrophoretic mobility. See
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