Abstract
Background: Ferrocene-containing β-diketones and cisplatin, [(NH3)2PtCl2], possess strong antineoplastic activity. No information is available regarding the anticancer activity of the corresponding ruthenocene complexes. This study examined the cytotoxicity of stable ruthenocene-containing β-diketones. The results were related to the cytotoxicity of cisplatin and the ease of ruthenium electrochemical oxidation. Materials and Methods: The ruthenocene-containing β-diketones RcCOCH2COR where Rc=RuII(C5H5)(C5H4) and R=CF3 (1), CH3 (2), Ph=C6H5 (3) and Fc=FeII(C5H5)(C5H4) (4) were tested for cytotoxicity against HeLa (human cervix epithelioid) cancer, COR L23 (human large cell lung carcinoma) and the platinum-resistant CoLo 320DM (human colorectal) and COR L23/CPR cancer cell lines. Cell survival was measured by means of the colourimetric 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay. Results: The 50% cell growth inhibition (IC50) values of 1-4 towards the cells ranged between 8.2 and 84.6 μmol dm−3, with 1 being the most cytotoxic complex. Drug activity was directly proportional to the electron density on the ruthenium centre as well as the oxidation potential of the ruthenium core but inversely proportional to the pKa of the β-diketones. The strongest activity was observed against the COR L23 cell line, and the weakest activity against COR L23 CPR. Conclusion: A drug activity–structural relationship exists for ruthenocene-containing β-diketones in that drugs with the lowest electron density on the ruthenium centre are more cytotoxic. Compounds with larger ruthenium oxidation potentials and stronger acid strength (i.e. smaller pKa values) are more cytotoxic.
High excretion rates from the body, the development of drug resistance after continued drug dosage, high toxicity, especially to the kidneys and bone marrow, and, perhaps most importantly of all, the inability to distinguish between healthy and carcinomatous cells, are some of the negative side-effects that cisplatin (1, 2) and many other chemotherapeutic drugs suffer from. As a consequence of this, new antineoplastic agents are continuously being synthesised and evaluated (3, 4), combinatorial therapies are being investigated in the hope of finding synergistic effects (5), new methods of delivering an active drug to a malignant growth are developed (6-8) and new techniques for cancer treatment, such as photodynamic cancer therapy (9), are being investigated.
It has been shown that free ferrocene-containing β-diketones (10) are more active than cisplatin against platinum resistant CoLo 320DM (human colorectal) and COR L23/CPR (human large lung cell) cancer cell lines. In contrast, carboxylato complex of ruthenium (11) were found to be two to four orders of magnitude less active than cisplatin.
In this study, we determined and compared the in vitro cytotoxicity of ruthenium-containing β-diketones 1-4 and diruthenium tetrakisacetate, 5. The relationship between cytotoxicity and β-diketone acid strength expressed as pKa values (12), ruthenium oxidation potentials, Epa (12), and electron density on the ruthenium centre expressed as a function of group electronegativities of the R group, χR (13), are also reported.
Materials and Methods
Compounds. Complexes 1-4 (Figure 1) were synthesised according to published procedures (12). Cytotoxic results for 5 and cisplatin under the same conditions were described elsewhere (8, 11).
Sample preparation. The samples were dissolved in dimethyl sulphoxide (DMSO) giving stock concentrations of 20 mmol dm−3 and diluted in the appropriate growth medium supplemented with foetal calf serum (FCS) to give final DMSO concentrations not exceeding 0.5% and drug concentrations of 20-2,000 μmol dm−3 prior to the cell experiments.
Cell cultures. The human colorectal cell lines, CoLo 320DM (ATCC CCL-220) (American Type Culture Collection, Manassas, VA, USA), was grown as a suspended culture in RPMI-1640. The human cervix epitheloid cancer cell line, HeLa (ATCC CCL-2) (American Type Culture Collection), human large cell lung carcinoma cell line, COR-L23 (ECACC 92031919) (European Collection of Animal Cell Cultures, Salisbury, Witshire, UK) and a cisplatin-resistant sub-line, COR-L23/CPR (ECACC96042336) (European Collection of Animal Cell Cultures), were grown as monolayer cultures using Eagle's minimum essential medium (MEM) in the case of HeLa cells and RPMI-1640 in the case of the other cell lines. The growth media were maintained at 37°C under 5% CO2 and fortified with 10% FCS, and 1% penicillin and streptomycin. The cells were seeded at 400 cells/well in 96 well microtiter plates in a final volume of 200 μl of growth medium in the presence or absence of different concentrations of the experimental drugs. Appropriate solvent control systems were included.
Structure of ruthenocene-containing β-diketones 1-4 and the ruthenium carboxylate 5. Ph=phenyl=C6H5, Fc=ferrocenyl=FeII(C5H5) (C5H4).
In accordance with previous studies (10), incubation at 37°C was allowed for 7 days before cell survival was measured by means of the colourimetric 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazodium bromide (MTT) assay (14). The plates were read on a spectrophotometer at 570 nm with a reference wavelength of 630 nm. Wells without cells and with cells but without drugs were included as controls. Survival curves were plotted (Figure 2) as a function of drug dose and the drug concentration that caused 50% inhibition of cell growth (IC50) was estimated by extrapolation.
Results
The cell growth-inhibitory properties of 1-5 are summarised in Table I. β-Diketone IC50 values ranged between 8.2 and 84.6 μmol dm−3. Lower IC50 values correspond to more active compounds. The most active drug was found to be the fluorinated β-diketone 1, while the COR L23/CPR cell line was the most resistant to the tested drugs. For this cell line, the cytotoxicity of complex 1 was the same as that of cisplatin, but for the other cell lines, cisplatin was slightly more cytotoxic than complex 1. RcCOCH2COCH3, 3, was up to 8 times less active than the CF3 complex 1 and the least cytotoxic in the β-diketone series. All β-diketones were much more cytotoxic than the carboxylate salt [Ru2(CH3COO)4.2H2O][PF6], 5. Complex 1 was two orders of maginitude (50-100 times) more cytotoxic than 5.
Effect of concentration of 1 (*), 2 (▪), 3 (●), and 4 (○) on the survival of COR L23 CPR cancer cells. Data are presented as the mean drug concentration±standard error of the mean of four experiments.
Discussion
In this study, the electron density on the ruthenium centre of the Rc group of β-diketones 1-4 was manipulated by introducing electron-withdrawing or electron-donating R substituents to the β-diketone RcCOCH2COR. It was previously shown that the electron density on the ruthenium core is a function of the electronegativity, χR, of each R group (12). Complex 1, which contained the strongest electron-withdrawing group, CF3 (χCF3=3.01), in its structure, is the complex with the electron-poorest ruthenium centre. This compound was also found to be the most cytotoxic (Table I). Because complex 4 with R of ferrocenyl has the strongest electron-donating R-group in its structure (χFc=1.87), the ruthenium centre of complex 4 should be the most electron-rich and therefore it should possess the lowest cytotoxicity of the compound series 1-4. However, the CH3 derivative 3 (χCH3=2.34) was less cytotoxic than 4, Table I. To account for this observation, the known high cytotoxicity of ferrocene-containing β-diketones is noted (10). It was concluded that a co-operative effect exists between the cytotoxic ferrocenyl and ruthenocenyl groups in 4 that overshadows any deactivation effect of the ruthenium centre that the electron-donating ferrocenyl group may induce. Since the CH3 group does not possess similar cytotoxic behaviour to the ferrocenyl group, complex 3 was found to be the least cytotoxic in the present β-diketone series. The cytotoxicity of the phenyl derivative, 2, was unexpectedly high and did not fit the cytotoxicity trend set by the other compounds.
The relationship between β-diketone acid strength, pKa, and cytotoxicity for HeLa cells is shown in Figure 3. From the observed trend, in the absence of any synergistic effects between the ferrocenyl and ruthenocenyl groups, the cytotoxicity of 4 would approach 100 μmol dm−3 or even higher rather than the observed 54.7 μmol dm−3. Although the ferrocenyl group increased the expected cytotoxicity of 4 by probably at least two-fold, this cytotoxic activation was still not sufficient for 4 to become more cytotoxic than the CF3 complex, 1.
Cytotoxicity of RcCOCH2COR complexes 1-4, the ruthenium carboxylate 5 and cisplatin expressed as IC50 (μmol dm−3) valuesa after 7 days of incubation with the indicated cancer cell lines.
pKa-cytotoxicity relationship for β-diketones RcCOCH2COR with R=CF3, Ph, CH3 or Fc against HeLa cells. R-groups are noted next to each data point. The broken line estimates the IC50 value of 4 if there was no synergistic effect between the ferrocenyl and ruthenocenyl groups. Complex 2 with R=Ph was not fitted to the line.
Figure 4 shows the relationship between ruthenium oxidation potentials, Epa, and compound cytotoxicity for CoLo 320DM cancer cells. The highest cytotoxicity (lowest IC50 value) is associated with the largest oxidation potential. From this relationship the enhanced cytotoxicity of β-diketone ruthenium compounds over carboxylato ruthenium compounds (11) can be explained. The carboxylato ligands cause the ruthenium centre to have a very low redox potential compared to that of the β-diketones. his practically eliminates any useful cytotoxic properties the ruthenium centre may possess, probably because the ruthenium centres become too deactivated to become involved in electron transfer reactions with key biological molecules such as DNA.
Relationship between oxidation potential and cytotoxicity of RcCOCH2COR and [Ru2(OOCCH3)4.2H2O][PF6], 5, against CoLo 320DM cells. Where relevant, R-groups are indicated next to each data point. Insert: β-diketone region only. Complex 2 with R=Ph was not fitted to the lines.
Conclusion
The cytotoxicity of RcCOCH2COCF3 and cisplatin, (H3N)2PtCl2, is comparable. Ruthenium-containing β-diketones are one to two orders of magnitude more cytotoxic than the corresponding ruthenium-containing acetate complex, 5. Substituents that induce lower electron density on the ruthenium centre, such as CF3, increase the cytotoxicity of the RcCOCH2COR series of compounds substantially. A synergistic effect between the ruthenocenyl and ferrocenyl groups in RcCOCH2COFc cause this complex to be more cytotoxic than expected. The cytotoxicity of RcCOCH2COR complexes is directly proportional to the oxidation potential of the ruthenium centre of the ruthenocenyl group but inversely proportional to β-diketone acid strength (pKa).
Acknowledgements
The National Research Foundation of South Africa under grant no. IFR2011040600072 and the Central Research Fund of the University of the Free State are acknowledged for their financial support.
- Received April 29, 2012.
- Revision received May 26, 2012.
- Accepted May 28, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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