Anti-colorectal cancer activity of an organometallic osmium arene azopyridine complex†
Steve D.
Shnyder
*a,
Ying
Fu
b,
Abraha
Habtemariam
b,
Sabine H.
van Rijt
b,
Patricia A.
Cooper
a,
Paul M.
Loadman
a and
Peter J.
Sadler
*b
aInstitute of Cancer Therapeutics, University of Bradford, Richmond Road, Bradford, BD7 1DP, UK. E-mail: S.D.Shnyder@Bradford.ac.uk; Fax: +44 (0)1274 233234; Tel: +44 (0)1274 235898
bDepartment of Chemistry, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK. E-mail: P.J.Sadler@warwick.ac.uk; Fax: +44 (0)24 76523819; Tel: +44 (0)24 76523818
First published on 19th May 2011
Abstract
This first in vivo antitumour activity for an organometallic osmium arene complex, [Os(η6-p-cym)(4-(2-pyridylazo)-N,N-dimethylaniline)I]PF6, is reported. The complex delays the growth of HCT116 human colon cancer xenografts in mice, with negligible toxicity. Its activity appears to involve redox mechanisms and its potency towards A2780 ovarian and A549 lung cancer cells is increased significantly in combination with L-buthionine-sulfoximine.
There is much current interest in exploring the anticancer activity of compounds containing metal–carbon bonds (organometallic compounds), especially those containing π-bonded aromatic cyclopentadienyl (C5 binding, five-fold hapticity, η5) or arene (C6 six-fold hapticity, η6) ligands.1 These include sandwich complexes of FeII and TiIV containing two π-bonded rings, and half-sandwich complexes of RuII and OsII, which contain just one π-bonded ring.2 The half-sandwich ‘piano-stool’ complexes have a particularly attractive scaffold for drug design since variations in the arene can modulate cell uptake and DNA intercalation, and the ligands which constitute the ‘legs’ of the ‘piano-stool’ can control the stability and reactivity. Initial cancer cell cytotoxicity data for ruthenium complexes of the type [Ru(η6-arene)(XY)Z]PF6 where, for example, the arene = biphenyl, XY = an N,N-chelated diamine such as ethylenediamine, and Z a leaving group such as Cl have shown activity against a range of types of cancer cells at low micromolar doses and no cross-resistance with cisplatin. Moreover some of these Ru arene complexes are active in vivo against human A2780 ovarian and A549 lung cancers.3,4 They have one reactive Ru–Cl bond and are thought to target DNA.5 However the biphenyl/ethylenediamine/Cl osmium analogue was subsequently found to be inactive in vivo in a mammary carcinoma model.6
The introduction of XY = azopyridine, a strong π-acceptor, as the N,N-chelating ligand has a remarkable effect on the reactivity, especially when Z = iodide.7 Both the RuII and the OsII arene azopyridine complexes are relatively unreactive, for example, being inert towards aquation. Recently we reported that the OsII azopyridine complex [Os(η6-p-cym)(azpy-NMe2)I]PF6, complex 1 (Fig. 1), is active in human A2780 and cisplatin-resistant A2780 ovarian, A549 lung, HCT-116 colon, MCF-7 breast, PC-3 prostate and RT-112 bladder cancer cell lines at sub-micromolar concentrations.8 For the HCT116 colon cancer cell line for example, the IC50 value is 220 nM.8 We have now investigated the activity of this complex in vivo versus HCT116 human colon cancer xenografts and the distribution of osmium in plasma and tissues. Further insight into the mechanism of action of 1 was obtained from studies of its redox potential, its ability to generate Reactive Oxygen Species (ROS) in cells, and the effect of L-buthionine-sulfoximine (L-BSO), a specific inhibitor of γ-glutamyl-cysteine synthetase known to reduce intracellular thiol levels,9 on the cytotoxicity of the complex.
 | ||
| Fig. 1 The structure of complex 1 (FY026). | ||
First we studied the anticancer efficacy of complex 1in vivo versus the subcutaneously implanted HCT-116 xenograft model, when administered as a single intravenous injection at its maximum soluble dose of 40 mg kg−1.‡ The agent had negligible toxicity, with an observed maximum weight loss well within the normal limits. Complex 1 was seen to induce a statistically significant tumour growth delay in the HCT-116 model compared to the untreated control (p < 0.01). The positive control compound, the standard agent cisplatin, was employed and administered i.p. (standard route for this model) at its MTD in this mouse model (p = 0.05, Fig. 2 and Table 1). The maximum weight loss observed with complex 1 was 8%, indicating a lack of toxicity. This combined with the favourable tumour distribution reported below would suggest that there is significant scope to administer this compound on a repeat-dose schedule to enhance its therapeutic activity.
 | ||
| Fig. 2 Evaluation of the in vivo efficacy of complex 1 when administered as a single intravenous injection at its maximum soluble dose of 40 mg kg−1 in the subcutaneously implanted HCT-116 human colon adenocarcinoma model. Complex 1 shows a greater efficacy than the standard agent cisplatin administered at its maximum tolerated dose in this model. Points represent mean ± S.D. (n = 8). | ||
| Compound (dose in mg kg−1) | Mean tumour doubling time/days | Significance | Maximum % weight loss (days) |
|---|---|---|---|
| Untreated controls | 3.9 | 8 (8) | |
| Complex 1 (40.0) | 6.5 | p < 0.01 | 8 (1) |
| Cisplatin (8.0) | 6.4 | p = 0.05 | 8 (6) |
Next we investigated the plasma and tissue distribution of osmium after administration of 1in vivo, in HCT116 xenograft-bearing mice. The distribution of 1 was analysed in the liver, kidneys, tumour, lungs and plasma, 5, 60 and 240 min after administration. The results are shown in Table 2 and Fig. S1†. Osmium was detected in the tumour and all tissues over the time period of analysis. The amount of osmium in the plasma was surprisingly low after just 5 min, suggesting a large volume of distribution or high level of tissue distribution.
| Time/min | μg Os per g tissue | ||||
|---|---|---|---|---|---|
| Kidney | Lungs | Liver | Plasma | Tumour | |
| 5 | 1259.7 (±66.6) | 5.81 (±4.18) | 88.6 (±57.9) | 19.6 (±23.3) | 11.9 (±2.22) |
| 60 | 513.5 (±208.5) | 40.3 (±15.0) | 136.4 (±4.96) | 21.8 (±2.54) | 8.80 (±1.24) |
| 240 | 418.6 (±103.3) | 28.3 (±3.49) | 77.2 (±10.5) | 19.0 (±10.6) | 6.68 (±2.77) |
The azopyridine complex 1 is relatively inert. For example, it does not readily undergo hydrolysis in aqueous solution or bind to DNA bases, unlike chlorido diamine OsII arene complexes.10 Our initial studies on iodido azopyridine OsII arene complexes8 suggested that their cytotoxic activity might involve redox mechanisms. Here we have investigated the ability of complex 1 to generate Reactive Oxygen Species (ROS) in cancer cells. ROS play important roles in regulating cell proliferation, death, and senescence. The redox system is also a significant target for anticancer treatment.11 Targeting the redox system can induce selective cell death in malignant cells and spare normal cells due to the higher baseline level of ROS in cancer cells.
We determined the level of ROS induced in A2780 human ovarian cancer cells induced by complex 1 using the probe 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH-DA). This is taken up by live cells, hydrolyzed to 2′,7′-dichlorodihydrofluorescein (DCFH), which in turn is oxidized to 2′,7′-dichlorofluorescein (DCF) in the presence of ROS and exhibits a green fluorescence.12 Using this probe, we determined the level of general oxidative stress induced in cells13 by 1. We also investigated the effect of combined exposure to 1 and L-buthionine-sulfoximine (L-BSO). L-BSO, a specific inhibitor of γ-glutamyl-cysteine synthetase, depletes intracellular glutathione (GSH). Glutathione plays a central role in a wide range of cellular functions, including protection, detoxification, transport, and metabolism.
A2780 cells were pre-incubated for 20 min with 10 μM DCFH-DA. The relative increase in DCF fluorescence was then detected over a period of 4 h after treatment of the cells with 1 μM 1, or 1 μM 1 plus 50 μM L-BSO, or 50 μM H2O2 (Fig. S2†). On treatment with 1 alone, the ROS level increased by 21% compared to the control. This level increased further to 50% in the presence of L-BSO in addition to 1. The mode of interference by 1 in the redox balance in cells and the production of ROS is not yet clear. Complex 1 underwent an electrochemical reduction in dimethylformamide with an associated half-wave potential of −0.64 V (vs.Ag/AgCl). This reduction can be associated with addition of an electron into the π* orbital centred on the azo group of the phenylazopyridine ligand to form an azo anion radical, a process previously detected for the RuII analog, although occurring more readily in the latter case at a potential of −0.40 V. Unlike the Ru complex,71 does not react catalytically with GSH.
These data suggested that L-BSO might enhance the cytotoxicity of complex 1 towards cancer cells. As can be seen from Fig. 3, L-BSO alone at a dose of 50 μM had no significant effect on the growth of either A2780 human ovarian cells or A549 human lung cancer cells, but greatly enhanced the cytotoxicity of complex 1 at doses of 0.1 and 1 μM for A2780 cells, and 1 and 5 μM for A549 cells. These data imply that combination treatment with 1 and L-BSO may have potential as a therapeutic strategy. Clinical trials of L-BSO are currently in progress on combinations with melphalan in patients with persistent or recurrent stage III malignant melanoma.14
 | ||
| Fig. 3 Percentage cell survival after 24 h exposure to osmium complex 1 (FY026) with or without 50 μM L-BSO. (Left) A2780 ovarian cancer cells; (Right) A549 lung cancer cells. Previously determined8IC50 values are 0.2 μM for A2780 cells (24 h exposure to 1 followed by 72 h recovery period) and 0.4 μM for A549 cells (24 h exposure, 96 h recovery period). | ||
In conclusion, we have demonstrated that the organometallic osmium arene azopyridine complex 1, which has nanomolar activity in vitro in a panel of human cancer cell lines,8 exhibits activity in vivo against HCT116 human colon cancer xenografts in mice, with negligible toxicity. This appears to be the first demonstration of significant anticancer activity in vivo for an organometallic half-sandwich osmium complex. Studies on the plasma, tumour and normal tissue distribution of 1 suggest that there is scope to optimize the therapeutic activity using multiple-dose schedules without the risk of off-target toxicity.
We thank the EPSRC (Knowledge Transfer Network), European Research Council (Award no. 247450) and Science City/EU ERDF/AWM for funding, Dr Ana Pizarro and Dr Michael Khan (University of Warwick) for provision of facilities for cell culture, Dr Michael Snowden and Professor Patrick Unwin (University of Warwick) for electrochemistry facilities, and members of COST Action D39 for stimulating discussions.
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Footnotes |
| † Electronic supplementary information (ESI) available: Materials, experimental procedures and Fig. S1 and S2. See DOI: 10.1039/c1md00075f |
| ‡ All in vivo experiments were carried out under a UK Home Office Project Licence (40/3133), which was granted following approval by the University of Bradford Ethical Review Panel. |
| This journal is © The Royal Society of Chemistry 2011 |