Behavior of the potential antitumor V^IVO complexes formed by flavonoid ligands. 3. Antioxidant properties and radical production capability

Highlights

• The antioxidant properties of V^IVO complexes formed by flavonoids were examined.

• The capability to reduce the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was studied.

• The capability to produce the ^•OH radical in Fenton-like reaction was described.

• The results were compared with those obtained with the aquaion [VO(H₂O)₅]²⁺.

• The results were also compared with [VO(acetylacetonato)₂] and [VO(catecholato)₂]²⁻.

Abstract

The radical production capability and the antioxidant properties of some V^IVO complexes formed by flavonoid ligands were examined. In particular, the bis-chelated species of quercetin (que), [VO(que)₂]^2 −, and morin (mor), [VO(mor)₂], were evaluated for their capability to reduce the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and produce the hydroxyl radical ^•OH by Fenton-like reactions, where the reducing agent is V^IVO^2 +. The results were compared with those displayed by other V^IVO complexes, such as [VO(H₂O)₅]²⁺, [VO(acac)₂] (acac = acetylacetonate) and [VO(cat)₂]^2 − (cat = catecholate). The capability of the V^IVO flavonoids complexes to reduce DPPH is much larger than that of the V^IVO species formed by non-antioxidant ligands and it is due mainly to the flavonoid molecule. Through the 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin trapping assay of the hydroxyl radical it was possible to demonstrate that in acidic solution V^IVO²⁺ has an effectiveness in producing ^•OH radicals comparable to that of Fe²⁺. When V^IVO complexes of flavonoids were taken into account, the amount of hydroxyl radicals produced in Fenton-like reactions depends on the specific structure of the ligand and on their capability to reduce H₂O₂ to give ^•OH. Both the formation of reactive oxygen species (ROS) under physiological conditions by V^IVO complexes of flavonoid ligands and their radical scavenging capability can be put in relationship with their antitumor effectiveness and it could be possible to modulate these actions by changing the features of the flavonoid coordinated to the V^IVO²⁺ ion, such as the entity, nature and position of the substituents and the number of phenolic groups.

Introduction

Over the last twenty years it has been demonstrated that vanadium compounds exhibit a wide variety of pharmacological properties both in animals and humans [1], [2]. The first possible application in medicine was in the therapy of diabetes [3], [4], [5]; bis(maltolato)oxidovanadium(IV) (BMOV) became the benchmark compound for the new molecules with anti-diabetic action [4], [6], [7], [8] and its derivative bis(ethylmaltolato)oxidovanadium(IV) (BEOV) arrived to phase IIa of the clinical trials [9], even if these have provisionally been abandoned due to renal problems arising with several patients [10], [11]. However, more recently, the research focused on other potential pharmacologically active compounds as anti-parasitic, spermicidal, anti-viral, anti-HIV and anti-tuberculosis agents [1], [10], [12] and, in particular, as antitumor drugs [12], [13], [14], [15].

The first report on the potential anticancer action of a vanadium compound, vanadocene dichloride (VDC), dates back to 1983 [16], [17]. Some years later, the anticancer activity of the compound known as Metvan, [V^IVO(4,7-Me₂phen)₂(SO₄)], where 4,7-Me₂phen is 4,7-dimethyl-1,10-phenanthroline, was demonstrated [18]. As discussed by Rehder, the organic ligand in a pharmacologically active V^IVO species should be non toxic at the physiological concentrations, cross the cell membranes, and form stable metal complexes easily absorbed in the gastrointestinal tract [19]. Recently the V^IVO complexes formed by flavonoids [20], [21], [22] are gaining increasing attention for their low cell toxicity and the resulting possibility to increase the administered concentration [23]. V^IVO species of flavonoid ligands have been demonstrated to be effective against osteosarcoma cell lines [24], [25], [26], [27], [28], [29], [30]; in particular, the bis-chelated V^IVO complex of quercetin (que) was shown to be more active than oxidovanadium(IV) ion and free quercetin [24] on tumoral osteoblast-like cells (UMR106). In addition, V^IVO complex of morin (mor) has been demonstrated to be a promising pharmacologically active substance against breast cancer (cells T47D and SKBR3) treatment [29].

However, the mechanism of action of vanadium compounds of flavonoids in the inhibition of cancer cell proliferation is still elusive [1]. As pointed out in the literature, the mode of action is certainly related to the speciation in the blood plasma [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], to the uptake of the compounds by the target cells and the subsequent biotransformation in the cell after the interaction with the cellular components [45], [46], [47], [48], [49], [50].

One of the properties of flavonoids which created more interest from the therapeutic point of view is the activity as antioxidants which may be exploited in the prevention of tumors. Therefore, the antioxidant properties of vanadium complexes with flavonoids could be related to their effect as antitumor agents. Indeed, DNA mutation is a critical step in carcinogenesis and elevated levels of oxidative DNA lesions (formation of 8-hydroxy-2′-deoxyguanosine, 8-OH-dG) have been noted in various tumors; the DNA damage is predominantly linked with the initiation step, in which the formation of reactive oxygen species (ROS) often occurs [51]. The antioxidant capacity of flavonoids is connected with the entity, nature and positions of the substituents and with the number of the phenolic groups [52]. In particular, a higher antioxidant activity can be observed when the B ring (Scheme 1) is the catechol moiety [52], since it can neutralize two radical molecules forming a stable quinone (Scheme 2a). Another important feature is the presence of a double bond in the position Δ^2,3 conjugated with the carbonyl group in position 4, and of an OH group in position 3 which increases the capability to neutralize radical species; the additional OH and/or OCH₃ groups in positions 5 and 7 seem to be less important. The role of the OH group in position 3 can be clearly observed in Scheme 2b, where the two electrons oxidation of morin is depicted. The oxidized form of morin is certainly less stable than that of quercetin but without the OH group in position 3, after the two electrons oxidation, it is possible to draw only unstable species with two unpaired electrons.

On these bases it can be argued that flavonols and flavons, having a B catecholic ring, are the most active species and that the flavonols (for example quercetin) are slightly more efficient than the corresponding flavons (for example luteolin) because of the presence of the OH group in position 3. Glycosylation of the phenol group in C-3 decreases a lot the capability to scavenge the radicals.

The antioxidant properties of the metal complexes can be complicated by the involvement of “non-innocent ligands”, such as catecholate derivatives. In fact when ligands containing the catechol moiety form a coordination compound with a metal ion, different possibilities exist: the ligand could be oxidized to semiquinone or quinone and the metal could be stabilized in a reduced form [53]. The oxidation of catechol derivatives to semiquinones is well-documented for some V compounds [54], [55] and the changes in the ligand structure were demonstrated recently by application of solid state NMR spectroscopy [56]. However, these processes do not occur in all the systems and, for example, with catechol, disodium 4,5-dihydroxybenzene-1,3-disulfonate (tiron), dopamine, epinephrine, norepinephrine and 3-(3,4-dihydroxyphenyl)alanine (L-dopa) redox reactions do not take place between V^IV and the ligand and the metal maintains its original oxidation state [57], [58], [59]. When the V species act as antioxidants, the oxidation process could take place at the expense of the metal ion, being oxidized to V^V, or of the ligand, forming semiquinone or quinone. Comparing the antioxidant properties of the complexes with those of the ligands, it is possible to say something about the role of the metal ion.

On the other hand, the cytotoxicity of vanadium complexes toward tumor cells can be related to the generation of reactive oxygen species (ROS) that would cause a series of cellular effects such as DNA cleavage and phosphotyrosine phosphatase (PTPase) inhibition (PTPase inhibition regulates different transduction pathways which trigger apoptosis, cell cycle arrest and modulation of proteins involved in the metastatic ability of the tumors) [12], [23], [60]. Therefore, as pointed out by Rehder, the participation of vanadium compounds in levelling ROS suggests that they can be beneficial in the treatment of several diseases and malfunctions related to ROS imbalances [19].

The generation of free radicals is strictly connected with the presence of a redox-active metal ion and the redox chemistry of vanadium compounds in the biological systems has been discussed by several authors [61], [62], [63], [64], [65], [66], [67], [68], [69]. In case of iron, homeostatic mechanisms guarantee that there is no free intracellular iron, which could participate to Fenton reaction generating the highly reactive hydroxyl radical:

Fe²⁺ + H₂O₂ → Fe³⁺ + ^•OH + OH^–

Other metal ions can give Fenton-like reactions such as Cu⁺, Ti³⁺ and Co²⁺, but also V^IV which is oxidized to V^V:

V^IVO²⁺ + H₂O₂ → V^VO₂⁺ + H⁺ + ^•OH

Vanadate(V) may be reduced by intracellular glutathione to vanadium(IV), as was recently confirmed in the erythrocytes [46], [47].

In this work the antioxidant properties of some V^IVO complexes formed by flavonoid ligands were examined; in particular, the bis-chelated species of morin (mor) and quercetin (que), recently studied by this group [70], [71], were evaluated. In principle, morin can form complexes with “maltol-like” and “acetylacetone-like” coordination, with the involvement of (CO, O^–) donor set and formation of five- or six-membered chelated rings. The geometry of the species formed could be cis-octahedral with a water molecule in the equatorial position and square pyramidal, respectively. The values of | A_x − A_y | = 7.0 × 10⁻⁴ cm⁻¹ and A_z = − 164.4 × 10⁻⁴ cm⁻¹, comparable with those of [VO(acac)₂], where acac is acetylacetonate (5.0 × 10⁻⁴ cm⁻¹ and A_z = − 166.5 × 10⁻⁴ cm⁻¹ [72]), suggested that the acetylacetone-like” coordination occurs (Scheme 1). Quercetin, beside the two binding modes available for morin, can form complexes with “catechol-like” coordination. At pH 7.4 a species is formed with A_z = − 155.7 × 10⁻⁴ cm⁻¹, very similar to that of [VO(cat)₂]^2 −, where cat is catecholate (A_z = − 155.6 × 10⁻⁴ cm⁻¹ [58]), and this demonstrated that quercetin binds V^IVO²⁺ ion with “catechol-like” donor set (O^–, O^–). Since (O^–, O^–) couple originates more stable complexes than the “acetylacetone-like” (CO, O^–) donor set, this is the preferred coordination at physiological pH (Scheme 1).

The capability to reduce the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and to produce the hydroxyl radical in Fenton-like reactions using the V^IVO²⁺ complexes as the reducing agents of hydrogen peroxide were measured with the DPPH assay and by trapping the hydroxyl radical with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), respectively. The results were compared with those observed in the systems containing inorganic V^IVO²⁺ ([VO(H₂O)₅]²⁺ ion), [VO(acac)₂] and [VO(cat)₂]^2 −. Since the vanadium(IV) complexes of flavonoids have a dual nature and on one hand they exhibit antioxidant properties, mainly due to the ligands, scavenging ROS and on the other they can participate to Fenton-like reactions generating hydroxyl radicals, it is not clear if their mechanism of action as antitumor agents is due to one of these two facts or to a combination of them. Therefore, the purpose of this paper is to evaluate these two aspects, comparing the properties of V^IVO-flavonoid complexes with those of the V species where the ligand is neither a flavonoid nor an antioxidant molecule.