Xin
Li
a,
Anil K.
Gorle
a,
Tracy D.
Ainsworth
b,
Kirsten
Heimann
*cd,
Clifford E.
Woodward
a,
J.
Grant Collins
*a and
F.
Richard Keene
*def
aSchool of Physical, Environmental and Mathematical Sciences, University of New South Wales, Australian Defence Force Academy, Canberra, ACT 2600, Australia. E-mail: g.collins@adfa.edu.au
bARC Centre of Excellence for Coral Reef Studies, James Cook University, Townsville, QLD 4811, Australia
cCollege of Marine & Environmental Sciences, James Cook University, Townsville, QLD 4811, Australia. E-mail: kirsten.heimann@jcu.edu.au
dCentre for Biodiscovery and Molecular Development of Therapeutics, James Cook University, Townsville, QLD 4811, Australia
eDepartment of Matter & Materials, College of Science, Technology & Engineering, James Cook University, Townsville, QLD 4811, Australia
fSchool of Chemistry and Physics, University of Adelaide, Adelaide, SA 5005, Australia. E-mail: richard.keene@adelaide.edu.au
First published on 21st October 2014
Confocal microscopy was used to study the intracellular localisation of a series of inert polypyridylruthenium(II) complexes with three eukaryotic cells lines – baby hamster kidney (BHK), human embryonic kidney (HEK-293) and liver carcinoma (Hep-G2). Co-staining experiments with the DNA-selective dye DAPI demonstrated that the di-, tri- and tetra-nuclear polypyridylruthenium(II) complexes that are linked by the bis[4(4′-methyl-2,2′-bipyridyl)]-1,12-dodecane bridging ligand (“bb12”) showed a high degree of selectivity for the nucleus of the eukaryotic cells. Additional co-localisation experiments with the general nucleic acid stain SYTO 9 indicated that the ruthenium complexes showed a considerable preference for the RNA-rich nucleolus, rather than chromosomal DNA. No significant differences were observed in the intracellular localisation between the ΔΔ and ΛΛ enantiomers of the dinuclear complex. Cytotoxicity assays carried out over 72 hours indicated that the ruthenium complexes, particularly the tri- and tetra-nuclear species, were significantly toxic to the eukaryotic cells. However, when the activity of the least cytotoxic compound (the ΔΔ enantiomer of the dinuclear species) was determined over a 24 hour period, the results indicated that the ruthenium complex was approximately a 100-fold less toxic to liver and kidney cells than to Gram positive bacteria. Circular dichroism (CD) spectroscopy was used to examine the effect of the ΔΔ and ΛΛ enantiomers of the dinuclear complex on the solution conformations of RNA and DNA. The CD experiments indicated that the RNA maintained the A-type conformation, and the DNA the B-type structure, upon binding by the ruthenium complexes.
More recently, due to the nucleic binding properties of inert polypyridylruthenium(II) complexes, there has been increasing interest in their biological properties.9–16 A variety of mononuclear and dinuclear complexes have shown good in vitro anticancer activity, which is generally considered to be due to DNA binding. However, in some cases other mechanisms of action have been proposed – e.g. interactions with membranes or mitochondrial-mediated apoptosis.15 In addition to the established anticancer properties of inert polypyridylruthenium(II) complexes, there is now growing recognition of their potential as antimicrobial agents. Antimicrobial resistance is an increasingly serious threat to global public health: infections caused by antibiotic-resistant bacteria are associated with increased morbidity and mortality.17 The lack of new antimicrobials in the pipeline to replace those in current use which are becoming ineffective has fostered research into the development of new types of drugs.
Dwyer and co-workers initially demonstrated the antimicrobial activity of mononuclear polypyridylruthenium(II) complexes against both Gram negative and Gram positive bacteria.18,19 We have subsequently shown that dinuclear analogues have even greater antimicrobial potential: [{Ru(phen)2}2{μ-bbn}]4+ {“Rubbn”; where phen = 1,10-phenanthroline; bbn = bis[4(4′-methyl-2,2′-bipyridyl)]-1,n-alkane for n = 5, 7, 10, 12 and 16 – see Fig. 1} showed excellent activity, and they maintained the activity against drug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA).20 Furthermore, preliminary toxicity assays against human red blood cells and a human white blood leukemia cell line (THP-1) demonstrated that the Rubbn complexes were not toxic to human cells at the concentrations required to kill the bacteria.20
While the affinity of polypyridylruthenium(II) complexes for nucleic acids can be readily demonstrated in vitro, it is more important to establish nucleic acid binding in live cells at concentrations similar to those required for anticancer or antimicrobial activities. Although there have been relatively few cellular localisation studies of polypyridylruthenium(II) complexes, the results reported to date have demonstrated a surprisingly diverse range of binding sites in eukaryotic cells. For example, Svensson et al. showed that the cellular localisation of a series of ruthenium dipyridophenazine (dppz) complexes in Chinese hamster ovarian cells was dependent upon the relative lipophilicity.21 The least lipophilic complex was predominantly found in the nucleus and the most lipophilic accumulated outside of the nucleus and probably in the endoplasmic reticulum. Furthermore, Gill et al. demonstrated that the DNA groove-binding dinuclear complex [{Ru(phen)2}2{μ-tpphz}]4+ (where tpphz = tetrapyridophenazine) could be used to image nuclear DNA in eukaryotic cells.11 Alternatively, the 4,7-diphenyl-1,10-phenanthroline analogue [{Ru(DIP)2}2{μ-tpphz}]4+ localised in the endoplasmic reticulum.22 By contrast, the Rubbn complexes were shown to localise in the mitochondria of L1210 white blood cells.12 Mitochondrial targeting has also been observed for other ruthenium complexes.15
As preliminary pharmacokinetic studies indicated that the Rubbn complexes accumulate in the liver and kidney of mice,23 we sought to confirm that the ruthenium complexes localised in the mitochondria of liver and kidney cells as we had previously demonstrated with the L1210 cells.12 In the present study, we examined the localisation of Rubb12 and its tri- and tetra-nuclear analogues in liver and kidney cells by confocal microscopy. In order to examine the effect of the ruthenium complexes on large DNA and RNA molecules, we also studied the binding of the ruthenium complexes to calf thymus DNA and baker's yeast RNA by CD spectroscopy. Interestingly, Rubb12 was found to selectively accumulate in the nucleolus, the RNA-rich component of the nucleus, rather than in the mitochondria.
The baker's yeast RNA (Sigma Aldrich) stock solution was also prepared in phosphate buffer in DEPC (Sigma Aldrich) treated water. The A260/A280 value was 2.10, indicating the RNA was pure.28 The initial RNA concentration was 2.3 mM (bases). The titration of ruthenium complexes and the data collection were the same as indicated for the DNA experiments.
Fig. 2 Structures of the trinuclear (Rubb12-tri) and tetranuclear (Rubb12-tetra) ruthenium(II) complexes. |
Table 2 shows the comparison of the IC50 values of the ruthenium complexes against the eukaryotic cells to the corresponding MIC values against the Gram positive bacterium S. aureus and the Gram negative species E. coli. Compared to the healthy eukaryotic BHK and HEK-293 cell lines, the ruthenium complexes exhibited a selectivity index (SI = IC50/MIC) of between 12 and 91 when compared to the Gram positive bacterium S. aureus, but only between 5 and 22 for the Gram negative E. coli. Interestingly, all ruthenium complexes were more toxic to the cancer cell line Hep-G2, and consequently they exhibited a lower SI value. Of the ruthenium complexes, ΔΔ-Rubb12 exhibited the best SI when compared to the healthy eukaryotic cell lines.
BHK | HEK-293 | Hep-G2 | ||||
---|---|---|---|---|---|---|
S. aureus | E. coli | S. aureus | E. coli | S. aureus | E. coli | |
ΔΔ-Rubb12 | 91 | 22 | 25 | 6 | 9 | 2 |
ΛΛ-Rubb12 | 40 | 19 | 12 | 6 | 8 | 4 |
Rubb12-tri | 53 | 13 | 22 | 6 | 19 | 5 |
Rubb12-tetra | 44 | 11 | 21 | 5 | 17 | 4 |
4 hour | 8 hour | 24 hour | 48 hour | 72 hour | |
---|---|---|---|---|---|
BHK | 190.9 ± 36.5 | 103.8 ± 8.5 | 70.5 ± 26.4 | 57.5 ± 7.1 | 54.3 ± 3.2 |
HEK-293 | 90.8 ± 17.9 | 90.48 ± 34.3 | 50.9 ± 19.9 | 24.9 ± 1.1 | 15.1 ± 2.8 |
Hep-G2 | 103.2 ± 3.8 | 109.7 ± 29.3 | 61.7 ± 5.5 | 15.8 ± 10.4 | 5.2 ± 2.0 |
The localisation in the nucleus was confirmed through co-staining with DAPI. DAPI is considered to be a DNA-selective stain, as it binds DNA 100-fold more strongly than RNA and has a 3-fold higher fluorescence quantum yield when bound to DNA than to RNA.29 In Fig. 4 we show the results of the DAPI co-staining experiments with ΔΔ-Rubb12 and BHK cells. The ΔΔ-Rubb12 concentration was 50 μM (approx. IC50) and the incubation time was 20 hours. These results confirm the preferential accumulation of the ruthenium complexes in the nucleus, however the localisation pattern was not identical. While significant DNA binding of the complex was observed at this concentration (as evidenced by the overlap with DAPI staining), there is also intense ΔΔ-Rubb12 red fluorescence in areas of the nucleus where there is little or no DAPI fluorescence. These so-called “DAPI holes” are generally recognised as nucleoli.30 The nucleolus is the site within the nucleus where ribosomal-RNA (r-RNA) is synthesised, and consequently is rich in r-RNA. The nucleoli can be highlighted through staining with SYTO 9. This general nucleic acid stain binds both DNA and RNA but binds RNA with greater affinity. The results of SYTO-9 co-staining experiments (also shown in Fig. 4) confirmed that ΔΔ-Rubb12 does accumulate in the nucleoli.
As is observed in Fig. 4, there is considerable DNA co-staining at 50 μM; however, at 10 μM there appears to be predominant RNA binding, and almost exclusive RNA binding at 5 μM (see Fig. 3).
Similar results were obtained with the other eukaryotic cells. For example, Fig. 5 shows the preferential accumulation of ΔΔ-Rubb12 in the nucleoli of Hep-G2 cells.
No significant difference in the localisation of the ΔΔ-Rubb12 and ΛΛ-Rubb12 enantiomers was observed. Similarly, for the Rubb12-tri and Rubb12-tetra complexes the same pattern of localisation was observed; however, the total accumulation appeared to be greater with more DNA binding observed for the Rubb12-tri and Rubb12-tetra complexes. Furthermore, increased accumulation was also observed outside of the nucleus (Fig. 6).
Fig. 6 Left to right – Rubb12-tetra localisation in BHK cells at 10 μM, stained by Mitotracker (green), Rubb12-tetra (red), DAPI (blue) and merged image. Scale bar = 10 μm. |
To examine the effect of time on the localisation of ΔΔ-Rubb12, BHK cells were incubated with ΔΔ-Rubb12 at 55 μM for both 4 and 20 hours. The resultant images are shown in Fig. 7. After a 4 hour incubation, ΔΔ-Rubb12 was localised to a greater extent in the cytoplasm compared to nucleolus. Subsequently, after the longer incubation time, the ΔΔ-Rubb12 was predominantly observed in the nuclear region, particularly in the nucleolus and nuclear envelope. These observations suggest that the ruthenium complexes will accumulate in the endoplasmic reticulum after passing through the cell membrane, but finally accumulate in the nucleolus. Similar results were obtained with ΛΛ-Rubb12 (data not shown).
Addition of both ΔΔ- and ΛΛ-Rubb12 induced significant decreases in the CD signal for the CT-DNA with added ruthenium complex in the 260–300 nm range at concentrations below the IC50 values (Fig. 8). However, and most clearly seen for the titration with ΛΛ-Rubb12, the basic B-type conformation is maintained (negative peak at 245 nm and positive peak at 260–280 nm). The decrease in intensity of the CD signal between 260 and 300 nm is consistent with the changes noted for the addition of high concentrations (5 M) of NaCl to CT-DNA32 – a decrease in the CD signal between 260 and 300 nm caused by high salt concentration is generally interpreted as the DNA structure becoming more tightly wound, but remaining in the B conformation.
Confocal microscopy was used to determine the cellular localisation of the ruthenium complexes in the three cell lines. By comparison with DAPI and SYTO 9 staining, it was concluded that Rubb12, Rubb12-tri and Rubb12-tetra preferentially accumulated in the nucleolus at low complex concentrations, while significant DNA binding is also observed at higher concentrations. The preference for RNA is consistent with our previous study on the localisation of ΔΔ-Rubb16 in the ribosomes of E. coli.37 The overall preference of these complexes to the nucleus is surprising given the mitochondrial selectivity we observed for the Rubbn complexes in L1210 cells.12 Also of note is the difference between the rigidly linked tetrapyridophenazine (tpphz) dinuclear ruthenium complexes studied by Thomas and co-workers11,22 and the flexibly-linked bbn complexes examined in this study. The less lipophilic [{Ru(phen)2}2{μ-tpphz}]4+ (logP = −0.96) targeted the nucleus, but not the nucleolus, and showed little toxicity towards MCF-7 cancer cells IC50 = 138 μM).11 On the other hand, the more lipophilic analogue containing the 4,7-diphenyl-1,10-phenanthroline ligand [{Ru(DIP)2}2{μ-tpphz}]4+ (logP = 1.52) targets the endoplasmic reticulum and is highly toxic to MCF-7 cells (IC50 = 7 μM).22 The bbn linked oligonuclear complexes are less lipophilic but all show greater toxicity to the cell lines studied than [{Ru(phen)2}2{μ-tpphz}]4+, and despite the differences in logP values, they all target the nucleolus. The results of this study suggest that in these cases logP values do not reflect the ease with which the ruthenium complexes can cross cell membranes. Although it is acknowledged that the [{Ru(phen)2}2{μ-tpphz}]4+ complexes enter cells by active transport,11 the results of this study suggest that the distance between the ruthenium centres (compared to the length of the highly non-polar section of a lipid bilayer) could be a more important factor for cellular uptake than lipophilicity, per se.
The CD spectroscopy experiments confirmed that Rubb12 can interact with DNA and RNA at biologically relevant concentrations. While the CD results suggested that Rubb12 affected the base-stacking of both DNA and RNA, there was no indication that the ruthenium complex condensed or aggregated either nucleic acid at ≤IC50 concentrations. Furthermore, both the RNA and DNA maintained their normal solution conformations upon ΔΔ/ΛΛ-Rubb12 binding. Given the preferential RNA binding exhibited by the ruthenium complexes, it is possible that RNA binding is responsible for the cellular toxicity. In support of this proposal is the observation that after 4 hours incubation with BHK cells, confocal microscopy indicated that a large proportion of the administered Rubb12 was located outside of the nucleus, but after 20 hours nearly all the ruthenium complex was inside the nucleus in the nucleolus. The IC50 value after a 4 hour incubation in the BHK cells was 190.9 μM, but this dropped to 70.5 μM after 24 hours and then only decreased to a small extent over the next 48 hours.
The results of this study indicate that the oligonuclear ruthenium complexes do bind nucleic acids in live cells, thereby supporting the proposed biological potential suggested in the many studies of nucleic acid binding by cationic transition metal complexes.1–8 However, it appears the ruthenium complexes target RNA rather than DNA. We have previously demonstrated that the bulky dinuclear ruthenium complexes bind in the DNA minor groove and preferentially target non-duplex features, such as bulges and hair-pin loops, compared to standard duplex structures.38,39 It could be argued that RNA contains a greater proportion of non-duplex structures than does DNA. However, generally only a slight difference was seen between the ΔΔ and ΛΛ enantiomers in terms of toxicity, intracellular localisation or in binding to long segments of DNA or RNA. In particular, a relatively larger enantiomeric effect is seen for Hep-G2 and S. aureus. As we have observed differences in the way the enantiomers interact with DNA oligonucleotides,7,38 it is possible that the effects observed in this study are primarily due to non-specific electrostatic interactions that cause sufficient structural modifications to inhibit RNA-driven transcription. The CD spectroscopy studies indicated that the ruthenium complex-bound DNA maintained the B-conformation, while the bound-RNA maintained the A-form. The A-form RNA has a shorter rise per base pair (≈2.8 Å) than B-DNA (≈3.4 Å).40 Consequently, A-RNA will have an increased linear negative charge density compared to B-form DNA. This should impact on the binding of polycations, and potentially when coupled to the increased proportion of more flexible non-duplex structures found in RNA, provides an explanation for the observed binding preference of the ruthenium complexes for RNA in the eukaryotic cells studied here.
In conclusion, the results of this study demonstrate that the Rubbn class of antimicrobial agents selectively accumulate in the nucleus of eukaryotic cells. However, the ruthenium complexes preferentially localise in the RNA-rich nucleoli, rather than with the chromosomal DNA. Although RNA and DNA binding is most likely responsible for the toxicity of the ruthenium complexes to the eukaryotic cells, the cytotoxicity assays indicated that the lead complex, ΔΔ-Rubb12, is approximately 100-fold less toxic to eukaryotic cells than to Gram positive bacteria.
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