Targeting acute myeloid leukemia cells with novel photosensitive ruthenium-based metal-organic compounds. (c2015)

Abstract

Acute myeloid leukemia (AML) is a class of hematopoietic malignancies that is characterized by the accumulation of abnormally differentiated myeloid cells and their infiltration into the bone marrow, blood and other tissues. Prognosis of patients diagnosed with AML remains low in all age groups, and most currently approved therapies against AML have serious side-effects and limited potency, necessitating the development of alternative, more potent approaches that could specifically target cancerous cells. Promising results have been reported in the field of Metallomics, in which various cancers have been targeted by compounds formed of different ligands conjugated to a metal core, one of which being ruthenium (Ru). Furthermore, some of these Ru complexes have been recognized for their photochemical potential. In this study, we sought to investigate the effects of bis-bidentate (precursor) and tris-bidentate (final) Ru(II) metal complexes, in which the ligands are phenanthroline- or bipyridine-derivatives, on a panel of human AML cell lines. We first established a structure-activity relationship of the precursors and their corresponding free ligands. Of the four different precursors tested {Ru-I: [Ru(II)(1,10-phenanthroline)2Cl2]; Ru-II: [Ru(II)(4,7-diphenyl-1,10-phenanthroline)2Cl2]; Ru-III: [Ru(II)(4,7-diphenyl-1,10-phenanthroline-disulfonate)2Na2]2+ and Ru-IV: [Ru(II)(2,2'-bipyridine)2Cl2]}, significant activity on seven out of nine cell lines, was detected only with Ru-II (IC50 values in the low μM range). Of the four free ligands tested (L-I, L-II, L-III and L-IV corresponding to Ru-I, Ru-II, Ru-III and Ru-IV, respectively), significant cytotoxic activity was detected in eight out of nine cell lines, only with L-I and L-II, (IC50 values in the low μM range for L-I and reaching the nM range for L-II). The activity of the free ligands L-I and L-II was at least one order of magnitude higher than that of the corresponding Ru(II) precursors. The activity of the metal complexes correlated with the structure of the ligands with modifications of phenanthroline (L-I) that make it more hydrophobic strongly increasing the activity of the free ligand itself (L-II), as well as that of the corresponding metal complex (Ru-II). The incorporation of a negatively-charged group (sulfonate) even to the more hydrophobic 4,7-diphenyl-1,10-phenanthroline, abolishes any activity in both the free ligand (L-III) and metal complex (Ru-III), suggesting that DNA intercalation is a possible mechanism of action of these compounds. No activity was detected with the ligand-free Ru(II) metal control, indicating that the activity of these Ru(II) metal complexes is not due to the metal center alone, but to the metal coordinated to ligand. The cytotoxic effect of L-II was shown to occur at earlier time points than that of Ru-II, although maximum effect for both was reached at 72h, possibly indicating a simpler mechanism of cytotoxicity in the ligand. Flow cytometric analysis of the uptake of Ru-II showed early uptake (< 1h post treatment), cell cycle analysis has shown G0-G1 cell cycle arrest at the 6h time-point with both L-II and Ru-II, an effect which was still observed with L-II at 24h, whereas a higher proportion of cell death was observed with Ru-II at 24h. Furthermore, we have attempted to examine the photochemical potential of our metal-organic compound model by combining L-II and L-III in the same metal complex, thus generating four final complexes with differing net charges (from +2 to -4), and examining their activities on a panel of AML cell lines with or without exposure to blue light. Of the four final complexes Ru-II3, Ru-II2-III, Ru-II-III2 and Ru-III3, only Ru-II3 and Ru-II2-III have shown significant activity against all tested cell lines in the dark, whereas photo-activation against ML2 has shown a marked increase of potency of all four complexes, even those shown to be biologically inert in the dark, reaching IC50 values in the nM range. The observed potentiation by light was of one order of magnitude for the compounds already showing potency in the dark, while reaching at least two orders of magnitude for those inactive in the dark. We hypothesize that the increased potency observed with all four complexes upon exposure to light might be due to a different mechanism, namely the release of Reactive Oxygen Species (ROS), thus affecting the cellular metabolism and viability through DNA damage, lipid peroxidation, amino-acid oxidation and oxidation of enzyme co-factors. In this study, we aim to establish a ruthenium-based model for effectively targeting AML, with the goal of later developing approaches to selectively target AML with complexes based on this model. We are still investigating the role of other ligands when conjugated to a ruthenium core, as well as characterizing the cellular mechanisms underlying the activity of these compounds.