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PERSPECTIVES:
Non-traditional platinum compounds for improved accumulation, oral bioavailability, and tumor
targeting
Katherine S. Lovejoy and Stephen J. Lippard, Dalton Trans., 2009, DOI: 10.1039/b913896j
Metal complexes as photochemical nitric oxide precursors: Potential applications in the treatment
of tumors
Alexis D. Ostrowski and Peter C. Ford, Dalton Trans., 2009, DOI: 10.1039/b912898k
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An oxo-bridged diruthenium(III) complex containing pyra- DNA,9 hexanuclear [Ru6 (p-iPrC6 H4 Me)6 (tpt)2 (dhbq)3 ]6+ (tpt =
zolato and pyrazole ligands is stable against ascorbic-acid 2,4,6-tris(pyridine-4-yl)-1,3,5-triazine, dhbq = 2,5-dihydroxy-1,4-
reduction, induces apoptosis (60%, 48 h) against HeLa cells at benzoquinonato),10 [RuIII (terpy)X3 ] (terpy = 2,2¢:6¢,6¢¢-terpyri-
10 lM level and exhibits promising anti-angiogenic activity at dine, X = Cl, Br or I),11 [RuIII (edta)(OH2 /OH)]1-/2- (edta =
its sub-cytotoxic concentrations. Other mononuclear ruthe- ethylenediaminetetraacetato),12 and Na2 [{trans-RuIII Cl4 -
nium(III) complexes containing pyrazole ligands [Ru(pz)4 X2 ]+ (Me2 SO)}2 (m-L)] (L = heterocyclic bridging ligand).13 Two
exhibit dual anti-angiogenic and cytotoxic properties. ruthenium-based anti-cancer drug leads, imidazolium[trans-
tetrachloro(1H -imidazole)(S-dimethylsulfoxide)ruthenate( III )]
Platinum(II) complexes as exemplified by cisplatin and its (NAMI-A) and indazolium[trans-RuCl4 (1H-indazole)2 ]
derivatives have been widely used for the treatment of various (KP1019), are separately anti-angiogenic and cytotoxic, res-
types of cancer.1 Despite their widespread clinical use, these pectively, and have entered phase I clinical trials.14,15
complexes suffer from severe toxic side effects and acquired drug Bioactive compounds with dual cytotoxic and anti-angiogenic
resistance.2 Thus, there are continuing efforts to search for new properties can enhance anti-cancer potency by reducing the
bioactive metal complexes including that of platinum,3 gold,4 acquired-drug resistance and systematic toxicities generated by
and ruthenium,5,6 as modern therapeutics. Notably, ruthenium using either a cytotoxic or an anti-angiogenic agent alone.16 Several
complexes have received considerable interest for this purpose in clinically-used cytotoxic compounds employed in metronomic
recent years.6 A wide range of ruthenium complexes have been chemotherapy (low doses on a continuous schedule) have recently
shown to be anti-tumour active, including RuII arene complexes been reported to display promising anti-angiogenic properties
[e.g., (h6 -arene)RuII (pta)Cl2 or [(h6 -arene)RuII (en)Cl]+ (pta = in vivo,17 though such examples are sparse in the literature. It
1,3,5-triaza-7-phosphaadamantane, en = ethylenediamine)],7 RuII has also been demonstrated that combination of an anti-cancer
pyridocarbazolyl complexes, which target the active site of large agent employed in transarterial chemoembolization (TACE) with
kinase,8 dinuclear RuII triple-stranded helicates that bind and coil an anti-angiogenic/metastatic agent can significantly prolong
survival of patients having advanced hepatocellular carcinoma
a
Department of Chemistry and Open Laboratory of Chemical Biology of (HCC).18a Notably, TACE involves the direct transportation of
the Institute of Molecular Technology for Drug Discovery and Synthesis, chemotherapeutic agents to the hepatic artery, the latter is the
The University of Hong Kong, Pokfulam Road, Hong Kong, China. E-mail: sole supplier of blood to the HCC tumour.18b Thus, an approach
cmche@hku.hk; Fax: (+852) 2857-1586
b
Department of Biology and Chemistry, City University of Hong Kong,
to a more efficient TACE treatment is to use compounds having
Tat Chee Avenue, Kowloon Tong, Hong Kong, China. E-mail: bhtclau@ dual cytotoxic and anti-angiogenic properties, which could induce
cityu.edu.hk apoptosis in high-density tumour region with local high-dose
† Electronic supplementary information (ESI) available: Experimental de- level, and induce anti-angiogenesis to cancer cells in adjacent or
tails; syntheses and characterization of complexes 1-5. UV-vis absorption
remote areas without damaging the surrounding normal cells or
spectrum of 1 in CH2 Cl2 (Fig. S1). UV-vis spectra of 1 in a PBS-EtOH
solution (Fig. S2). UV-vis spectra of 1 in a PBS-EtOH solution containing tissue. In this regard, we are interested to investigate whether
ascorbic acid (Fig. S3). UV-vis spectral changes of 1 in a PBS-EtOH cytotoxic ruthenium complexes can also have significant anti-
solution with increasing concentration of calf-thymus DNA (Fig. S4). angiogenic activity at their sub-cytotoxic levels. We report herein
Gel electrophoresis of 100-bp DNA in 2% (w/v) agarose gel showing the
mobilities of the DNA (15.2 mM bp-1 ) in the absence (first lane, left) and the ruthenium(III) pyrazolate complexes that exhibit dual cytotoxic
presence of ethidium bromide (EB), Hoechst33324 (H33324) and 1 (5 and and anti-angiogenic activities.
50 mM) (Fig. S5). Electrophoresis on agarose gel comparing the effect of The dinuclear ruthenium(III) complex (1, Ru2 (m2 -O)(m2 -
CPT and 1 on the relaxation plasmid DNA by human topoisomerase 1 pz)2 (pz)2 (pzH)4 where pz = pyrazolato and pzH = pyrazole)
(Fig. S6). UV-vis spectral changes of 1 a PBS-EtOH solution with
increasing concentration of HSA (Fig. S7). Detection of ROS generated was prepared by the reaction of Ru2 (OAc)4 Cl with pyrazole in
in the untreated and 1-treated HeLa cells (Fig. S8). Cytotoxicity of 1 MeOH–H2 O (1 : 1, v/v) at room temperature in 88% yield (ESI†).
toward MS1 cells (Fig. S9). Dose-dependent inhibition of the migration of Deep blue crystals of 1 were obtained by slow evaporation of a
MS1 cells by 1 (Fig. S10). Cyclic voltammogram of 1 recorded in CH2 Cl2
(Fig. S11). Cyclic voltammogram of 2 recorded in DMF (Fig. S12). Cyclic
CH2 Cl2 –hexane (1 : 2, v/v) solution. Complex 1 is stable at room
voltammogram of 3 recorded in DMF (Fig. S13). Cyclic voltammogram of temperature for months. By Evans NMR method,19 the complex is
4 recorded in DMF (Fig. S14). Inhibition of tube formation by 2, 3, 4 and diamagnetic. Its structure was characterized by X-ray crystallogra-
5 (Fig. S15). Cyclic voltammogram of 5 recorded in DMF (Fig. S16) and phy (Fig. 1).‡ Bond lengths and angles are given in the ESI (Table
bond lengths (Å) and angles (◦ ) for 1 (Table S1). CCDC reference number
687112. For ESI and crystallographic data in CIF or other electronic S1).† As depicted in Fig. 1, the complex contains two symmetry-
format see DOI: 10.1039/b912236b inequivalent Ru atoms (Ru1 and Ru2). The Ru1 and Ru2 atoms
10712 | Dalton Trans., 2009, 10712–10716 This journal is © The Royal Society of Chemistry 2009
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Since cancer is characterized by uncontrolled cellular prolifer- oxidant(s) upon treatment of HeLa cells with 1 (10 mM) and H2 O2
ation, there has been considerable interest in chemotherapeutic- (10 mM) for 0 to 5 h were examined using dihydrorhodamine-123
induced apoptosis.22 Using propidium iodide (PI) to assess the (DHRh-123) assay.24 By means of spectrophotometry, the fluo-
DNA content of cells, the apoptosis-inducing property of 1 in rescent signal of rhodamine-123+ (Rh-123+ ), which was generated
HeLa cells was examined by flow cytometry. Upon treatment with by the oxidation of non-fluorescent DHRh-123 in the presence
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1 (10 mM), 27.7% and 60% of HeLa cells were found to undergo of ROS, was monitored. We found that 1-treated HeLa cells
apoptotic cell death after 24 and 48 h incubation, respectively did not show any observable increase in fluorescent intensity of
(Fig. 4). Rh-123+ (Fig. S8, ESI†), revealing that the mechanism of action
was not due to the generation of intracellular ROS. Increasing
level of fluorescent signal (Rh-123+ ) was detected upon treatment
of the HeLa cells with H2 O2 only, but no fluorescent signal was
detected when the HeLa cells had been co-treated with both H2 O2
and 1 (Fig. S8, ESI).† These results reveal that the H2 O2 -induced
Rh-123+ formation is quenched by 1 implying that 1 is an effective
scavenger of ROS.
We further examined the inhibitory activity of 1 towards
nitric oxide (NO) production in lipopolysaccharide stimulated
endothelial cells (MS1). In this study, supernatants of MS1 cells
treated with 1 or vehicle control for 8 h were collected. The NO
present in the supernatants was oxidized to nitrate, and the total
nitrate content was subsequently quantified using a commercially
available NO assay kit.25 Compared to the vehicle control, the
8-h incubation of 1 (10 mM) significantly inhibited the total nitrate
formation by 28.1%, revealing that 1 could significantly inhibit NO
production in vitro. Since a number of ruthenium complexes have
long been known to display promising anti-angiogenic activity by
inhibiting nitric oxide formation,14,15 the anti-angiogenic activity
of 1 has also been investigated.
The initial step of angiogenesis involves endothelial cell
proliferation.26 Under in vitro conditions, endothelial cells are able
to form capillary-like structure on a layer of matrigel, a crude
extract of basal membrane from mouse Engelbreth-Holm-Swarm
Fig. 4 Induction of cell-cycle arrest in HeLa cells after treatment with 1.
tumours, which can be employed as a cell-base model for studying
Untreated cells and cells treated with 1 (10 mM) were collected at different
possible anti-angiogenic activities of different compounds. In this
time points (24 or 48 h), subjected to propidium iodide (PI) staining and
flow cytometric analysis. G1-phase cells (Dip G1), S-phase cells (Dip S),
work, tube-formation assay (8 h) for testing the anti-angiogenic
G2-phase cells (Dip G2) and apoptotic cells (apoptosis). activity of 1 has been performed. The maximum concentration
of 1 was restricted to 5 mM (Fig. S9, ESI†) in order to maintain
Cell-cycle-arrest study was also undertaken. As shown in Fig. 4, over 95% viability of the endothelial cells after 8 h treatment.
there is an apparent G1-phase cell-cycle arrest, resulting in a As depicted in Fig. 5, formation of capillary-like structure of the
concomitant decrease in the G2-phase population (from 33 to endothelial cells at 8 h was inhibited by 1, with 24% and 76%
~2%). On the other hand, no apparent S-phase cell-cycle arrest was inhibition at concentration of 1.25 mM and 2.5 mM, respectively.
observed upon treatment of HeLa cells with 1 for 48 h. Since DNA
damaging compounds such as cisplatin significantly cause S-phase
cell-cycle arrest and consequently inhibit DNA replication,23 this
finding reveals that 1 could not halt DNA replication in HeLa
cells under the experimental conditions. The direct interaction of
1 with DNA has been examined. We found that 1 weakly interacts
with calf-thymus DNA (ctDNA) with a binding constant of (6.3 ±
0.8) ¥ 103 dm3 mol-1 based on the results from spectrophotometric
titration experiments (Fig. S4, ESI).† Complex 1 also fails to
intercalate DNA (Fig. S5, ESI†) and has no inhibitory activity on
a DNA-binding protein topoisomerase I (Fig. S6, ESI).† Taken
these results altogether, DNA may not be a crucial cellular target of
1 in inducing its cytotoxicity. Besides DNA and topoisomerase I,
1 also displays weak interaction with human serum album (HSA)
with binding constant of (1.2 ± 0.2) ¥ 104 dm3 mol-1 at 298 K
(Fig. S7, ESI).†
A major anti-cancer mechanism involves the generation of reac- Fig. 5 The anti-angiogenic activity of 1 as revealed from the tube-forma-
tive oxygen species (ROS). In this study, intracellular production of tion assay (arrow: selected sites for tube formation).
10714 | Dalton Trans., 2009, 10712–10716 This journal is © The Royal Society of Chemistry 2009
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Notably, incubation of 1 at 5 mM led to >95% distortion of tube that a higher Ru uptake [cellular Ru content: 3 (19.8 nM) > 1 (17.1
formation. nM) > 2 (10.1 nM)] led to a higher cytotoxic activity (IC50 values:
Wound-healing-migration is an established protocol that allows 3 < 1 < 2). We further prepared and examined the cytotoxicity
examination of cell migration in response to an artificial wound of cis-[RuII (BiIml)2 (DMSO)2 ](ClO4 )2 (5, BiIml = biimidazole),29
produced on a cell monolayer.27 Complex 1 showed wound- in which case the ruthenium ion is stable in the +2 oxidation
Published on 14 September 2009. Downloaded by Heinrich Heine University of Duesseldorf on 29/11/2013 09:23:55.
healing capability on MS1 cells in a dose-dependent manner state with considerably higher E ◦ (RuIII/II ) value of +0.45 V vs.
(Fig. S10, ESI).† A distinct cell migration in the wound area Cp2 Fe+/0 (Fig. S16, ESI).† Similar to 4, this complex displays low
was found for the vehicle-control group 8 h after wounding, NO inhibitory activity (10.9% vs. control) and is neither cytotoxic
whereas the 1-treated cells at concentrations of 1.25, 2.5 and 5 mM (IC50 value > 100 mM) nor anti-angiogenic.
demonstrated dose-dependent inhibitory effects on wound healing We have demonstrated that a diruthenium(III) complex (1)
under similar conditions. Taken the findings altogether, complex displays dual cytotoxic and anti-angiogenic activities. These dual
1 displays promising in vitro anti-angiogenic effect at sub-toxic properties have also been found in two mononuclear mono-
concentrations. cationic ruthenium(III) complexes having pyrzaole ligands and
In order to investigate whether the dual cytotoxic and anti- with comparable E ◦ [RuIII /RuII ] values as that of 1 (E ◦ 1/2 = -0.61 V
angiogenic properties also present in other ruthenium complexes vs. Cp2 Fe+/0 ). In contrast, the two stable di-cationic ruthenium(II)
with pyrazole or 3-methylpyrazole ligands, complexes 2–4 with complexes 4 and 5 having comparatively high E ◦ [RuIII /RuII ]
structures depicted in Fig. 6 were prepared.28 Their electrochem- values do not display these biological activities.
ical, cytotoxic and anti-angiogenic properties were examined and
compared to that of 1. The cyclic voltammogram of 1 reveals two
reversible couples at E ◦ 1/2 = -0.61 V and -0.96 V vs. Cp2 Fe+/0 , Notes and references
which are assigned to the RuIII,III /RuIII,II and RuIII,II /RuII,II couples, ‡ Crystal data: 1·H2 O·(hexane)0.5 : C108 H148 N64 O8 Ru8 , M = 3279.5, tetrago-
respectively (Fig. S11, ESI).† For complexes 2, 3 and 4, a reversible nal, I41 cd, a = 29.596(1) Å, c = 15.253(1) Å, V = 13360.4(6) Å3 , T = 100 K,
RuIII /RuII couple is observed at E ◦ 1/2 = -0.40 V, -0.54 V and Z = 4, m = 7.764 mm-1 , 39 710 collected reflections, 5635 independent
reflections [Rint = 0.038], data/restraints/parameters = 5635/90/444.
+0.097 vs. Cp2 Fe+/0 , respectively (Fig. S12–S14, ESI).† Goodness of fit = 1.038. Final R indices: R1 (I > 2s) = 0.059, wR2 =
0.150.
This journal is © The Royal Society of Chemistry 2009 Dalton Trans., 2009, 10712–10716 | 10715
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13 E. Alessio, E. Iengo, S. Zorzet, A. Bergamo, M. Coluccia, A. Boccarelli Chem. Commun., 2000, 1547; (c) A. J. Blake, N. R. Champness, A. N.
and G. Sava, J. Inorg. Biochem., 2000, 79, 173. Khlobystov, S. Parsons and M. Schröder, Angew. Chem., 2000, 112,
14 E. Alessio, G. Mestroni, A. Bergamo and G. Sava, Curr. Top. Med. 2407, (Angew. Chem., Int. Ed., 2000, 39, 2317).
Chem., 2004, 4, 1525. 22 (a) A. Hunt and G. Evan, Science, 2001, 293, 1784; (b) M. O.
15 (a) M. Galanski, V. B. Arion, M. A. Jakupec and B. K. Keppler, Curr. Hengartner, Nature, 2000, 407, 770.
Pharm. Des., 2003, 9, 2078; (b) C. G. Hartinger, S. Zorbas-Seifried, 23 C. K.-L. Li, R. W.-Y. Sun, S. C.-F. Kui, N. Zhu and C.-M. Che, Chem.–
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M. A. Jakupec, B. Kynast, H. Zorbas and B. K. Keppler, J. Inorg. Eur. J., 2006, 12, 5253.
Biochem., 2006, 100, 891. 24 E. L.-M. Wong, G.-S. Fang, C.-M. Che and N. Zhu, Chem. Commun.,
16 G. Gasparini, R. Longo, M. Fanelli and B. A. Teicher, J. Clin. Oncol., 2005, 4578.
2005, 23, 1295, and references therein. 25 J. Sun, X. Zhang, M. Broderick and H. Fein, Sensors, 2003, 3, 276.
17 R. S. Kerbel and B. A. Kamen, Nat. Rev. Cancer, 2004, 4, 423, and 26 R. Auerbach, R. Lewis, B. Shinners, L. Kubai and N. Akhtar, Clin.
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18 (a) H. Graf, C. Jungst, G. Straub, S. Dogan, R.-T. Hoffmann, T. Jakobs, 27 M. Li, S. S.-M. Ng, J. Wang, L. Lai, S. Y. Leung, M. Franco, Y. Peng,
M. Reiser, T. Waggershauser, T. Helmberger, A. Walter, A. Walli, D. M.-L. He, H.-F. Kung and M. C.-M. Lin, Cancer Res., 2006, 66, 1583.
Seidel, B. Goke and D. Jungst, Digestion, 2008, 78, 34; (b) W. Y. Lau, 28 The synthesis of 2 has recently been reported. E. Reisner, V. B. Arion, A.
S. C. Yu, E. C. Lai and T. W. Leung, Journal of the American College Eichinger, N. Kandler, G. Giester, A. J. L. Pombeiro and B. K. Keppler,
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19 D. F. Evans, J. Chem. Soc., 1959, 2003. 29 The X-ray crystal structure of the cation of 5 has been reported in the
20 D. S. Bohle and E. S. Sagan, Eur. J. Inorg. Chem., 2000, 1609. Ph.D thesis of L. Shek, City University of Hong Kong, Hong Kong,
21 (a) G. Li, W. Yu and Y. Cui, J. Am. Chem. Soc., 2008, 130, 4582; (b) A. 2007. This complex has a distorted octahedral geometry. Two DMSO
Fragoso, M. L. Kahn, A. Castiñeiras, J.-P. Sutter, O. Kahn and R. Cao, ligands are S-bonded to metal center with a cis geometry.
10716 | Dalton Trans., 2009, 10712–10716 This journal is © The Royal Society of Chemistry 2009