Selective mitochondrial accumulation of cytotoxic dinuclear polypyridyl ruthenium(II) complexes

Abstract

The lipophilic ligand-bridged dinuclear cation Rubb₁₆ is significantly cytotoxic and preferentially accumulates in the mitochondria of the L1210 murine leukemia cancer cell line.

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It has been more than fifty years since Dwyer and coworkers showed that mononuclear tris(bidentate)ruthenium(II) complexes containing polypyridyl ligands have antibacterial activity.¹ Subsequent studies have shown that this class of compounds also has significant anticancer potential,^2–6 where enhanced cytotoxicity is correlated with increased lipophilicity. The mechanism of action for these complexes has generally been assumed to involve DNA interaction, although other mechanisms have been suggested.^5,6 Over the last decade there has been growing interest in inert dinuclear polypyridyl ruthenium(II) complexes, particularly as nucleic acid binding agents.^7,8 To date, dinuclear polypyridyl ruthenium(II) complexes have only exhibited modest levels of cytotoxicity;^9,10 however, it is probable that their cytotoxic properties could be significantly improved by increasing the overall lipophilicity as observed for the dinuclear ruthenium(II) arene complexes reported by Mendoza-Ferri et al.¹¹ We have been interested in the DNA binding ability of a series of dinuclear complexes where the metal centres are linked by a flexible bridge⁸ in which the lipophilicity can be systematically varied by increasing the number of methylene groups in the chain. In the present communication, we demonstrate that increasing the length of the alkane chain does significantly improve the cytotoxic properties. More interestingly, we show for the first time that dinuclear ruthenium(II) complexes can selectively accumulate in the mitochondria of live cancer cells and that for this series of complexes the level of accumulation is reflected in their cytotoxities.

We have studied a series of dinuclear ruthenium(II) complexes, ΔΔ/ΛΛ-[{Ru(phen)₂}₂{μ-bb_n}]⁴⁺ {“Rubb_n”; where phen = 1,10-phenanthroline; bb_n = 1,n-bis[4(4′-methyl-2,2′-bipyridyl)]-nane (n = 2, 5, 7, 10, 12 or 16; Fig. 1}, which only differ in the number of methylene groups in the bridging ligand between the two metal centres. Rubb_n (n = 2, 5, 7, 10) were obtained as their chloride salts as previously described,¹² while the Rubb₁₂ and Rubb₁₆ analogues were stereoselectively synthesised by the same method, but using 1,12-bis[4(4′-methyl-2,2′-bipyridyl)]dodecane and 1,16-bis[4(4′-methyl-2,2′-bipyridyl)]hexadecane as the linking ligands, respectively. Confirmatory NMR, elemental analysis, circular dichroism and mass spectroscopy data were obtained. The cytotoxicity of this family of ruthenium complexes was analysed in the L1210 murine leukemia cancer cell line. The results are summarised in Table 1. The complex Rubb₂ showed very low cytotoxicity, while that of Rubb₇ and Rubb₁₀ was relatively modest and similar to that reported for other inert dinuclear ruthenium(II) polypyridyl complexes:^9,10 however, Rubb₁₂ and Rubb₁₆ exhibited significant cytotoxicity to the leukemia cell line. Interestingly, the ΔΔ isomers of the more active ruthenium complexes (Rubb₁₂ and Rubb₁₆) were slightly more cytotoxic than their ΛΛ counterparts.

Fig. 1 The chemical structure of Rubb_n (where n = 2, 5, 7 10, 12 or 16 methylene groups in the linking chain).

Table 1 Cytotoxicity (IC₅₀) of Rubb_n in the L1210 murine leukemia cancer cell line. IC₅₀ is defined as the concentration of complex (μM) required to inhibit cell growth by 50%. IC₅₀ values were determined from at least two independent experiments. Errors were calculated from the standard deviation of the data set

Complex	IC50/μM
	ΔΔ		ΛΛ
a Comparative data on same L1210 cell line supplied by the Peter MacCallum Cancer Centre.
Rubb2	>200		>200
Rubb5	180 ± 42		158 ± 12
Rubb7	82 ± 12		100 ± 7
Rubb10	42 ± 7		50 ± 1
Rubb12	18 ± 10		23 ± 5
Rubb16	5 ± 1		15 ± 1
Carboplatin^a		5
Cisplatin^a		1

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Cellular uptake of the Rubb_n analogues can be compared using fluorescence microscopy.¹³ As shown in Fig. 2, all three Rubb_n complexes (n = 5, 10, 16) entered the L1210 cells. No obvious decrease in fluorescence intensity was observed, indicating that no significant quenching took place as a consequence of the ruthenium complexes binding to intracellular components or being chemically degraded. Incubation with 50 μM Rubb₅ resulted in luminescence inside the cell which was appreciably above the background, with sites of localisation visible. The luminescence intensity inside the cell increased dramatically with incubation of Rubb₁₀ and Rubb₁₆ at 50 μM, resulting in high fluorescence obscuring localisation patterns; however, incubation at 25 μM and 5 μM for Rubb₁₀ and Rubb₁₆, respectively, decreased the luminescence intensity to the point where organelle staining was visible (Fig. 2). Organelle staining for all metal complexes was suggestive of mitochondrial localisation.

Fig. 2 Fluorescence microscopy images of cellular uptake and organelle accumulation of Rubb_n. Incubation concentrations are: (a) Rubb₅ (50 μM), (b) Rubb₁₀ (25 μM) and (c) Rubb₁₆ (5 μM).

Confocal microscopy has been shown to be a useful tool for studying the cellular localisation of luminescent ruthenium(II) compounds.¹⁴ As the results of fluorescence microscopy studies suggested that mitochondria were the cellular sites of uptake for the Rubb_n complexes, confocal microscopy was used in conjunction with a mitochondrial tracking dye to identify organelle localisation sites within the cell. Localisation studies were undertaken by adding Mitotracker^® Green FM (Invitrogen) to the cell suspension during the last 30 min of incubation to give a final incubation concentration of 100–200 nM. Live cells were washed, mounted and viewed at 60× (oil) magnification on a BIO-RAD Radiance 2000 laser-scanning confocal microscope. Both the metal complex (λ_ex ∼ 450 nm, λ_em ∼ 610 nm) and Mitotracker (λ_ex ∼ 490 nm, λ_em ∼ 516 nm) were excited using a blue argon laser (488 nm). After four hours incubation, the site(s) of accumulation of the metal complexes were visible. For Rubb₅, the cellular staining indicated that the drug was significantly taken up by mitochondria (Fig. 3a); however other sites of accumulation were also observed. These sites are thought to be lysosomes and further studies are being undertaken to confirm this hypothesis. In contrast, all the Rubb₁₀ and Rubb₁₆ accumulated in the mitochondria, even at high complex concentrations, with no staining being observed in the cytoplasm or other organelles (Fig. 3b), consistent with observations at 5 μM.

Fig. 3 Confocal microscopy images of (a) Rubb₅ (50 μM) and (b) Rubb₁₆ (50 μM). Image (i) shows cellular staining of Rubb_n, (ii) Mitotracker^® Green FM staining, and (iii) a micrograph overlay showing sites of co-localisation. Areas stained yellow are indicative of drug localisation in mitochondria. Mitotracker^® Green FM incubation concentrations were 200 nM and 100 nM for (a) and (b), respectively.

Differential interference contrast light microscopy was used to determine cell characteristics in order to establish morphological changes induced by the addition of the metal complexes. The ruthenium complexes did induce several changes to the cell morphology, most notably the appearance of a grainy texture, blebbing of the plasma membrane, and irregular cell size and shape (Fig. 4). Cells incubated with Rubb₁₆ exhibited a greater degree of morphological changes compared to Rubb₅ and Rubb₁₀, most significantly a 20% reduction in the average cell size compared to untreated cells (Table 2) and an increase in the incidence of membrane blebbing.

Fig. 4 Differential interference contrast light micrographs of L1210 murine leukemia cells (left) untreated and (right) treated with Rubb₁₆ (5 μM) highlighting cell morphology changes induced by drug uptake; i.e. irregular cell shape and size, increased membrane blebbing and grainy texture.

Table 2 Minimum, maximum and average cell diameters of L1210 murine leukemia cells incubated with 50 μM Rubb_n (where n = 5, 10 or 16) and untreated cells. Values were derived from a data set of at least 150 cells for each metal complex and untreated control

	Cell diameter/μm
	Untreated	Rubb5	Rubb10	Rubb16
a Standard Error = Standard Deviation/(number of cells – 1)^1/2.
Minimum	19.8	19.2	11.4	13.1
Maximum	38.0	46.4	29.1	39.4
Average	26.8	26.5	19.4	20.3
Standard Error^a	±0.3	±0.6	±0.4	±0.5

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While Rubb₇ and Rubb₁₀ only showed modest cytotoxicity, similar to that reported for other inert dinuclear ruthenium(II) polypyridyl complexes,^9,10 Rubb₁₂ and Rubb₁₆ exhibited significant cytotoxicity to the leukemia cell line. It is concluded that the additional methylene groups in Rubb₁₂ and Rubb₁₆ increase the lipophilicity of the metal complex, thereby allowing greater cellular accumulation. DNA binding is often considered as a dominant mechanism of biological activity; indeed, Gill et al.¹⁵ reported that a dinuclear ruthenium(II) polypyridyl complex can be used to image DNA in living cells. However other mechanisms, e.g. modification of cell membrane function and cell adhesion, have been proposed.^5,6 Furthermore, while Puckett and Barton suggested some mitochondrial uptake, inter alia, for a mononuclear intercalating complex,¹⁴ this present study shows that the mitochondria are the predominate site of accumulation for our dinuclear complexes. Further experiments are under way to establish the precise mechanism of the cytotoxicity.

As the ruthenium complexes are localised in the mitochondria it is also concluded that they are acting as delocalised lipophilic cations. Owing to the fact that mitochondria play an important role in cell function—most notably energy generation, apoptosis, cell cycle control and calcium homeostasis¹⁶—it represents a key target for anticancer therapeutics. Consequently, lipophilic dinuclear ruthenium(II) complexes may have potential as anticancer agents.

Acknowledgements

This work was funded by the Australian Research Council. The L1210 murine leukemia cells were provided by the Peter McCallum Cancer Center, Melbourne, Australia and we thank Dr Carleen Cullinane for her cooperation and assistance. We thankfully acknowledge the technical assistance of Dr Kevin Blake (Advanced Analytical Centre, James Cook University) with the confocal microscopy experiments. We are also thankful to Professor Alan Baxter (Comparative Genomic Center, James Cook University) for the use of cell culture facilities, and Mr Martin Keene for assistance with the establishment of the cell line.

References

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