Antitumor activity of dinitrosyl iron complexes with glutathione

The antitumor dose-dependent effect of binuclear dinitrosyl iron complexes with glutathione as NO donors on a solid tumor in the mouse, Lewis lung carcinoma, was detected. The complexes being injected at doses of 21, 42, 105 mg/kg daily for 10 days blocked completely the development of the tumor for the first week after tumor cell implantation into animals. After that, the part of tumor cells which remained in intact alive state began to grow at a rate equal to that for control animals. The effect was proposed to be caused via formation of an antinitrosative defense system in the cells as a response to NO attack on cells. It was also hypothesized that this system can be inactivated by higher doses of dinitrosyl iron complexes. Data were obtained which were in line with the hypothesis.     

The cytoprotective effect of nitrite is based on the formation of dinitrosyl iron complexes

Nitrite protects various organs from ischemia–reperfusion injury by ameliorating mitochondrial dysfunction. Here we provide evidence that this protection is due to the inhibition of iron-mediated oxidative reactions caused by the release of iron ions upon hypoxia. We show in a model of isolated rat liver mitochondria that upon hypoxia, mitochondria reduce nitrite to nitric oxide (NO) in amounts sufficient to inactivate redox-active iron ions by formation of inactive dinitrosyl iron complexes (DNIC). The scavenging of iron ions in turn prevents the oxidative modification of the outer mitochondrial membrane and the release of cytochrome c during reoxygenation. This action of nitrite protects mitochondrial function. The formation of DNIC with nitrite-derived NO could also be confirmed in an ischemia–reperfusion model in liver tissue. Our data suggest that the formation of DNIC is a key mechanism of nitrite-mediated cytoprotection.     

The antitumor effect of dinitrosyl iron complexes with glutathione in a murine solid-tumor model

A significant antitumor activity of aqueous solutions of binuclear dinitrosyl iron complexes with glutathione was found when they were injected intravenously in a model of a solid malignant tumor, that is, Lewis carcinoma, in mice. Dinitrosyl iron complexes completely inhibited the tumor growth (by 100%) at doses of 20, 10, and 2 μmol/kg in the first 11 days after the beginning of experiment followed by tumor proliferation at a rate that was lowest for the lowest of the used doses. At day 16, the inhibition of tumor growth was 90% when a solution of dinitrosyl iron complexes was injected at a dose of 2 μmol/kg five times with an interval of 2 to 3 days between injections; whereas the inhibition of tumor growth did not exceed 70 and 30% at doses of 10 and 20 μmol/kg, respectively. Acceleration, rather than inhibition of carcinoma growth was observed at a dose of 100 μmol/kg. The tumor weight increased 1.5–2.0 times compared to the control values, depending on the time.

     

Formation of dinitrosyl iron complexes in cardiac mitochondria

It has been established that, in the presence of S-nitrosothiols, cysteine, and mitochondria, dinitrosyl iron complexes (DNIC) coupled to low-molecular-weight ligands and proteins are formed. The concentration of DNIC depended on oxygen partial pressure. It was shown that, under the conditions of hypoxia, the kinetics of the formation of low-molecular DNIC was biphasic. After the replacement of anaerobic conditions of incubation with aerobic ones, the level of DNIC came down; in this case, protein dinitrosyl complexes became more stable. We proposed that iron-and sulfur-containing proteins and low-molecular-weight iron complexes are the sources of iron for DNIC formation in mitochondrial suspensions. It was shown that a combination of DNIC and S-nitrosothiols inhibited effectively the respiration of cardiomyocytes.     

Beneficial effect of dinitrosyl iron complexes with thiol ligands on the rat penile cavernous bodies

Dinitrosyl iron complexes (DNIC) with thiol ligands were found to beneficially affect the state of the penile cavernous tissue upon its experimental denervation in rats. Histological and histochemical analysis showed that intracavernous administration of DNIC (twice weekly over six months) almost completely abolished the proliferation of endothelial cells typical of denervated cavernous tissue. On the other hand, this treatment sustained the mitotic activity of smooth myocytes and prevented the appearance of collagenase, a marker of their fibrotic transformation. The DNIC treatment had a pronounced effect on penile erection in neurotomized as well as in intact animals. Introduction of low-molecular DNIC into cavernous tissue was found to cause formation of protein-bound complexes observed by EPR and probably acting as depots of nitric oxide, ensuring steady erection.     

L-cysteine-mediated destabilization of dinitrosyl iron complexes in proteins.

Nitric oxide is a signaling molecule in intercellular communication as well as a powerful weapon used by macrophages to kill tumor cells and pathogenic bacteria. Here, we show that when Escherichia coli cells are exposed to nitric oxide, its ferredoxin [2Fe-2S] cluster is nitrosylated, forming the dinitrosyl iron complex with a characteristic EPR signal at g(av) = 2.04. Such formed ferredoxin dinitrosyl iron complex is efficiently repaired in E. coli cells even in the absence of new protein synthesis. However, the repair activity is completely inactivated once E. coli cells are disrupted, indicating that repairing the ferredoxin dinitrosyl iron complex requires cellular reducing equivalents. In search of such cellular factors, we find that l-cysteine can effectively eliminate the EPR signal of the ferredoxin dinitrosyl iron complex and release the ferrous iron from the complex. In contrast, N-acetyl-l-cysteine and reduced glutathione are much less effective. l-Cysteine seems to have a general function, since it can also remove the otherwise stable dinitrosyl iron complexes from proteins in the cell extracts prepared from the E. coli cells treated with nitric oxide. We propose that l-cysteine is responsible for removing the dinitrosyl iron complexes from the nitric oxide-modified proteins into which a new iron-sulfur cluster will be reassembled.     

Physicochemistry of dinitrosyl iron complexes with thiolate ligands underlying their beneficial effect in endometriosis

Exogenous dinitrosyl iron complexes (DNIC) with thiolate ligands as NO and NO⁺ donors are capable of exerting both regulatory and cytotoxic effects on diverse biological processes similarly to those characteristic of endogenous nitric oxide. Regulatory activity of DNIC (vasodilatory, hypotensive, suppressing thrombosis, increasing erythrocyte elasticity, accelerating skin wound healing, inducing penile erection, etc.) is determined by their capacity of NO and NO⁺ transfer to biological targets of the latter (heme- and thiol-containing proteins, respectively) due to higher affinity of the proteins for NO and NO⁺ than that of DNIC. Cytotoxic activity of DNIC is provided by rapid DNIC decomposition under action of iron-chelating compounds, resulting in appearance of NO and NO⁺ in cells and tissues in high amounts. The latter mechanism is suggested to cause the blocking effect of DNIC as cytotoxic effectors on the development of benign endometrial tumors in rats with experimental endometriosis. It is also proposed that a similar mechanism can operate to cause at least a delay of malignant tumor proliferation under action of DNIC.     

Distribution and pharmacokinetics of dinitrosyl iron complexes in rat organs

Dinitrosyl iron complexes (DNICs) have been traced in rat blood and organs after intravenous infusion of Oxacom. It is shown that the active principle (DNIC with glutathione) is rapidly distributed through the organism and deposited in blood and organs as protein-bound DNICs. The specific levels of DNIC in the main body organs are comparable, whereas its apparent lifetimes relate as blood < heart = lung < liver < kidney. Spin trapping assays indicate that protein-bound DNICs are a major but not the only form of NO deposition; the next largest depot is most probably formed by S-nitrosothiols. The gradual release of NO from such pools ensures the smooth and prolonged hypotensive effect of Oxacom.     

Vasorelaxing activity of stable powder preparations of dinitrosyl iron complexes with cysteine or glutathione ligands.

Vasorelaxant activity of new stable powder preparations of dinitrosyl iron complexes (DNIC) with thiol-containing ligands was investigated on rat abdominal aorta rings. The preparations preserve their physicochemical characteristics (EPR and optical absorption) if stored for a long time in dry air (at least half-year). Three preparations of DNIC were tested: diamagnetic dimeric DNIC with glutathione (DNIC-GS 1:2) or cysteine (DNIC-cys 1:2) and paramagnetic monomeric DNIC with cysteine (DNIC-cys 1:20). Being dissolved in physiological solution the preparations induced relaxation of vessel similarly to that by earlier described non-stable DNICs which should be stored in liquid nitrogen. The amplitudes and kinetic characteristics of the relaxation were dependent on the incorporated thiolate ligands. Rapid transient relaxation followed by significant tone recovery to stationary level (plateau) was observed for DNIC-cys 1:2. DNIC-cys 1:20 also induced initial rapid relaxation followed by incomplete tone recovery. DNIC-GS 1:2 induced slow developing and long lasting relaxation. NO scavenger, hydroxocobalamin (2x10(-5)M) eliminated the rapid transitory relaxation induced by DNIC-cys 1:20 and did not influence significantly on the plateau level. SOD increased duration of the DNIC-cys 1:2 and DNIC-cys 1:20 induced relaxation. The addition of 5x10(-5)M DNIC-cys 1:2 or DNIC-cys 1:20 induced long lasting vasorelaxation within 20min and more. However the EPR measurements demonstrated full rapid disappearance (within 1-2min) of both type of DNIC-cys in Krebs medium bubbled with carbogen gas. This was not the case for DNIC-GS 1:2. We suggested that the long lasting vasorelaxation observed during the addition of DNICs-cys was induced by S-nitrosocysteine derived from DNICs-cys and stabilized by EDTA in Krebs medium. The suggestion is in line with the fact that strong ferrous chelator bathophenantroline disulfonate (BPDS) which is capable of rapid degradation of DNICs did not abrogate the vasorelaxtion induced by DNIC addition.     

Crucial role of lysosomal iron in the formation of dinitrosyl iron complexes in vivo

Dinitrosyl non-heme–iron complexes (DNIC) are found in many nitric oxide producing tissues. A prerequisite of DNIC formation is the presence of nitric oxide, iron and thiol/imidazole groups. The aim of this study was to investigate the role of the cellular labile iron pool in the formation of DNIC in erythroid K562 cells. The cells were treated with a nitric oxide donor in the presence of a permeable (salicylaldehyde isonicotinoyl hydrazone) or a nonpermeable (desferrioxamine mesylate) iron chelator and DNIC formation was recorded using electron paramagnetic resonance. Both chelators inhibited DNIC formation up to 50% after 6 h of treatment. To further investigate the role of lysosomal iron in DNIC formation, we prevented lysosomal proteolysis by pretreatment of whole cells with NH₄Cl. Pretreatment with NH₄Cl inhibited the formation of DNIC in a time-dependent manner that points to the importance of the degradation of iron metalloproteins in DNIC formation in vivo. Fractionation of the cell content after treatment with the nitric oxide donor revealed that DNIC is formed predominantly in the endosomal/lysosomal fraction. Taken together, these data indicate that lysosomal iron plays a crucial role in DNIC formation in vivo. Degradation of iron-containing metalloproteins seems to be important for this process.     

Prospects of designing medicines with diverse therapeutic activity on the basis of dinitrosyl iron complexes with thiol-containing ligands

A stable hypotensive preparation (Oxacom) based on dinitrosyl iron complexes (DNIC) with glutathione has been developed. The preparation has successfully passed pharmacological trials. Tests on volunteers have shown a high hypotensive activity of the preparation: a single intravenous infusion of its aqueous solution at a dose of 0.2 μmol active substance per kg body wt led to a 20–30% decrease in arterial pressure, which persisted for 15–20 h. Similar experiments on animals demonstrated that aqueous solutions of DNIC with cysteine or glutathione also exert a hypotensive action due to their vasodilatory activity. Besides, these complexes accelerate wound healing and produce a potent erectile effect. There is reason to suppose that DNIC with thiol ligands as NO donors may be cytotoxic for pathogenic mycobacteria Mycobacterium tuberculosis and, after appropriate treatment, inhibit cancer cell proliferation. These complexes can be used as analgesics, for inhibiting the adhesion process, in treating preeclampsia, spermatogenesis pathologies, and in cosmetology for treatment of skin injury.     

Interaction between dinitrosyl iron complexes and intermediate products of oxidative stress

The interaction between glutathione-containing dinitrosyl iron complexes and superoxide radicals has been studied under the conditions of superoxide radical generation in mitochondria and in a model system xanthine-xanthine oxidase. It has been shown that both superoxide radical and hydroxyl radical are involved in the destruction of dinitrosyl iron complexes. At the same time, iron contained in dinitrosyl iron complex, apparently, does not catalyze the decomposition of hydrogen peroxide with the formation of hydroxyl radical. It has been found that dinitrosyl iron complexes with different anion ligands inhibit effectively the formation of phenoxyl probucol radical in a hemin-H2O2 a system. In this process, different components of the dinitrosyl iron complexes take part in the antioxidant action of these complexes.     

Effect of physico-chemical factors and species specificity on the structure of dinitrosyl iron complexes

Formation in mouse, rat and man's blood of iron nitrosyl complexes with pair thiol groups of proteins (complexes 2.03) was shown by ESR method. This formation was initiated by the introduction in blood in vitro or in vivo of low molecular dinitrosyl complexes of iron with phosphate, thiosulphate, cysteine or reduced gluthatione. Three forms of these complexes were found. They were characterized by ESR signals with rhombic or axial symmetry of g-factor tensor. These forms pass into one another under the effect of a number of thiol-containing compounds or at blood freezing. The life time of the complexes 2.03 in the blood in vivo is several hours.     

Prospects of designing the medicines with diverse therapeutic activity on the basis of dinitrosyl iron complexes with thiol-containing ligands

A stable hypotensive preparation (Oxacom) based on dinitrosyl iron complexes (DNIC) with glutathione has been developed. The preparation has successfully passed through pharmacological trials. The tests on volunteers have shown a high hypotensive activity of the preparation: a single intravenous infusion of its aqueous solution at a dose of 0.2 microM per kg of body weight led to a 20-30% decrease in arterial pressure, which persisted for a period of 15-20 h. Similar experiments on the animals demonstrated that aqueous solutions of DNIC with cysteine or glutathione exert also the hypotensive action due to their vasodilatory activity. Besides, these complexes accelerate wound healing and produce a potent erective action. There is reason to suggest that DNIC with thiol-containing ligands as NO donors can produce the cytotoxic action on the pathogenic mycobacteria Mycobacterium tuberculosis and, after respective treatment, inhibit cancer cell proliferation. These complexes can be used as analgetics, for inhibiting the adhesion process, in the therapy of preexplampsia, spermatogenesis pathologies, and in cosmetology for the treatment of skin injury.     

Inhibition of platelet aggregation by dinitrosyl iron complexes with low molecular weight ligands

The dinitrosyl iron complexes (DNIC) with thiosulphate, cysteine or phosphate were shown to inhibit in vitro (in citrate plasma) the human platelet aggregation induced by ADP, collagen or adrenaline. This effect cannot be explained by the toxic action of DNIC on the platelet membrane, since DNIC-pretreated platelets are capable of aggregating under the action of 10(-8) M/ml of phorbol ester, which is known to cause direct activation of protein kinase C. The antiaggregatory activity of DNIC exceeds that of Na-nitroprusside and seems to be due to nitric oxide capable to activate guanylate cyclase of platelets. Using the EPR method, it was shown that addition of DNIC to platelet-enriched plasma results in a rapid transfer of Fe(NO)2 groups to the coupled RS(-)-groups proteins of plasma and, apparently, of platelet membrane proteins. These protein DNIC seem to be the source of NO which inhibits human platelet aggregation.     

Physicochemical study of floranol, its copper(II) and iron(III) complexes, and their inhibitory effect on LDL oxidation

The antioxidant activity of floranol (3,5,7,2'-tetrahydroxy-6-methoxy-8-prenylflavanone), a new flavonoid isolated from the roots of Dioclea grandiflora, was evaluated by the inhibition of human low-density lipoprotein (LDL) oxidation. Floranol increased its oxidation lag-phase significantly in a dose-dependent manner. As the antioxidant mechanism may involve metal coordination, we have undertaken a detailed study of floranol interactions with Cu(II) and Fe(III) by combination of UV-visible (UV-Vis) and mass spectrometries and cyclic voltammetry. The acidity constants of the ligand as well as the stability constants of the metal complexes were calculated. The pKa values of 6.58, 11.97 and 13.87 were determined and the following acidity order is proposed 7-OH>5-OH>2'-OH. The best fit between experimental and calculated spectra was obtained assuming the formation of two Cu(II) complexes: [CuL] logbeta=19.34+/-0.05 and [CuL(2)](2-) logbeta=26.4+/-0.10 and three Fe(III) complexes: [FeL(3)](3-) logbeta=44.72+/-0.09, [FeL(2)](-) logbeta=35.32+/-0.08 and [FeL](+) logbeta=19.51+/-0.04. In addition, copper and iron reduction is less favorable in the presence of floranol. These results indicate that floranol can efficiently bind Cu(II) and Fe(III) ions thus preventing their effect on LDL oxidation.     

Effect of dinitrosyl iron complexes with thiol-containing ligands on the penile cavernosum bodies in rats

A beneficial effect of dinitrosyl iron complexes (DNIC) with thiol-containing ligands on penile cavernus tissue was shown in rats subjected to penile denervation. Histological and histochemical investigations demonstrated that intracavernous injections of dinitrosyl iron complexes (2 times per one week during 6 months) blocked the reinforcement of endothelial cell proliferation in the tissue characteristic of the cavernous tissue when the penile nerve was removed. On the other hand, treatment with dinitrosyl iron complexes led to the preservation of mitotic activity of smooth myocytes and protected against the appearance in these cells of collagenase, an indicator of muscle transformation into fibrous tissue. It was shown that the process of fibrous transformation of myocytes correlates with a decrease in the mitotic activity of fibroblasts in the adventive part of cavernosa. The mitotic activity increased in cavernous tissue in the absence of dinitrosyl iron complexes. The efficiency of long-term action of dinitrosyl iron complexes on the erection in both intact animals and animals subjected to neuroectomy of cavernous tissue nerve was shown. The injection of low-molecular dinitrosyl iron complexes to the cavernous tissue resulted in the formation of protein-bound dinitrosyl iron complexes in the tissue, which were detected by the EPR technique. It is assumed that these dinitrosyl iron complexes function as a depot of nitric oxide, providing long-lasting penis erection.     

The antitumor activity of the S-nitrosoglutathione and dinitrosyl iron complex with glutathione: Comparative studies

The antitumor activity of the binuclear form of dinitrosyl iron complexes with glutathione against Lewis lung carcinoma was found earlier with intraperitoneal administration of the complexes. This activity was also observed when this preparation was injected subcutaneously. The complex inhibited the tumor growth by 43% upon subcutaneous injection at a daily dose of 100 µM/kg (as calculated per one iron atom in the binuclear dinitrosyl iron complex) for 10 or 15 days. The effect was observed during the first 2 weeks after tumor transplantation. After this, the tumors began to grow at a rate that was equal to or even higher than that for the control animals. The mean survival time for the treated mice exceeded the control values by 30%. Binuclear dinitrosyl iron complexes were also effective against Ca-755 adenocarcinoma with intraperitoneal administration. In this case, however, the mean survival time for the treated animals only increased by 7%. It was also shown that S-nitrosoglutathione inhibited the growth of Lewis lung carcinoma and Ca-755 adenocarcinoma by 70 and 90%, respectively. However, in contrast to binuclear dinitrosyl iron complexes, the antitumor effect of S-nitrosoglutathione decreased with an increase in the daily dose of the compound from 200 to 400 µM/kg. The initial antitumor effect of binuclear dinitrosyl iron complexes and S-nitrosoglutathione is suggested to be due to NO that is released from both compounds. The subsequent suppression of the effect is caused by the activation of antinitrosative and antioxidant defense systems in tumors.     

The nitric oxide-iron interplay in mammalian cells: transport and storage of dinitrosyl iron complexes.

Nitrogen monoxide (NO) is a vital effector and messenger molecule that plays roles in a variety of biological processes. Many of the functions of NO are mediated by its high affinity for iron (Fe) in the active centres of proteins. Indeed, NO possesses a rich coordination chemistry with this metal and the formation of dinitrosyl-dithiolato-Fe complexes (DNICs) is well known to occur intracellularly. In mammals, NO produced by activated macrophages acts as a cytotoxic effector against tumour cells by binding and releasing cancer cell Fe that is vital for proliferation. Glucose metabolism and the subsequent generation of glutathione (GSH) are critical for NO-mediated Fe efflux and this process occurs by active transport. Our previous studies showed that GSH is required for Fe mobilisation from tumour cells and we hypothesized it was effluxed with Fe as a dinitrosyl-diglutathionyl-Fe complex (DNDGIC). It is well known that Fe and GSH release from cells induces apoptosis, a crucial property for a cytotoxic effector like NO. Furthermore, NO-mediated Fe release is mediated from cells expressing the GSH transporter, multi-drug resistance protein 1 (MRP1). Interestingly, the glutathione-S-transferase (GST) enzymes act to bind DNDGICs with high affinity and some members of the GST family act as storage intermediates for these complexes. Since the GST enzymes and MRP1 form a coordinated system for removing toxic substances from cells, it is possible to hypothesize these molecules regulate NO levels by binding and transporting DNDGICs.     

Physicochemical features of dinitrosyl iron complexes with natural thiol-containing ligands underlying the biological activities of these complexes

Current notions and new experimental data of the authors on physicochemical features of mono- and binuclear dinitrosyl iron complexes (DNIC) with natural thiol-containing ligands (glutathione or cysteine), underlying the ability of DNIC to act as NO molecule and nitrosonium ion donors, are considered. This ability determines the various biological activities of DNIC: inducing long-lasting vasodilation and thereby long-lasting hypotension in human and animals, inhibiting platelet aggregation, increasing red blood cell elasticity, thereby stimulating microcirculation, and reducing the necrotic zone in animals with myocardial infarction. Moreover, DNIC are capable of accelerating skin wound healing, improving the function of penile cavernous tissue, and blocking apoptosis development in cell cultures. When decomposed, DNIC can exert a cytotoxic effect that may be used in treatment for infection and malignant pathologies.