Antioxidant,DNA cleavage,and cellular effects of silibinin and a new oxovanadium(IV)/silibinin complex

A new complex of the oxovanadium(IV) cation with the flavolignan silibinin has been synthesized and characterized. Vanadium compounds show interesting biological and pharmacological properties and some of them display antitumoral actions. Flavonoids are part of a larger group of antioxidant compounds called polyphenols which may inhibit the proliferation and growth of cancer cells. The antioxidant and antitumoral effects of silibinin and its oxovanadium(IV) complex were investigated. Silibinin acted as a very strong antioxidant and its complexation with oxovanadium(IV) improved this behavior. Besides, the generation of reactive oxygen species (ROS) by this compound was favored in tumoral (UMR106) cells and correlated with the deleterious behavior in the proliferation of this cell line. Conversely, silibinin did not exert any effect on the proliferation of normal osteoblasts (MC3T3E1). The cytotoxic action and ROS generation of the oxovanadium(IV) complex was more effective in tumoral cells. This behavior was not consistent with cleaving DNA of plasmid DNA pA1 because no significant cleaving activity was observed in both cases. These results suggest that the main deleterious mechanisms may take place through cytotoxic effects more than genotoxic actions. A comparison with our own findings on the behavior of other flavonoids and their vanadyl(IV) complex has also been performed.     

Vanadyl(IV) complexes with saccharides. Bioactivity in osteoblast-like cells in culture

Complexes of vanadyl(IV) with 4 monosaccharides and 5 disaccharides were tested in 2 osteoblast-like cell lines (MC3T3E1 and UMR106). Many complexes caused stimulation of UMR106 proliferation (120% basal) in the range of 2.5 to 25 micromol/L. In the nontransformed osteoblasts, some vanadyl-saccharide complexes stimulated the mitogenesis (115% basal) in the same range of concentration. The glucose and sucrose complexes were the most efficient inhibitory agents (65% and 88% of inhibition vs. basal, respectively) for tumoral cells at 100 micromol/L. The galactose and turanose complexes exerted a similar effect in the nontransformed osteoblasts. On the other hand, all the complexes promoted the phosphorylation of the extracellular regulated kinases (ERKs). All together, these results indicate that the stimulation of ERKs is not the only factor that plays a role in the proliferative effects of vanadium derivatives since some compounds were inhibitory proliferating agents. Cell differentiation was evaluated by alkaline phosphatase specific activity and collagen synthesis in UMR106 cells. All the complexes inhibited alkaline phosphatase activity, with galactose complex as the most effective compound (IC50 = 43 micromol/L). The complex with the trehalose TreVO was the most effective agent to stimulate collagen synthesis (142% basal) and glucose consumption (132% basal). A cytosolic tyrosine protein kinase and the kinase-3 of glycogen synthase seem to be involved in the stimulation of glucose consumption by vanadium derivatives. In this series, only TreVO gathered the characteristics of a good insulin mimetic and osteogenic drug. In addition, this complex was a good promoting agent of nontransformed osteoblast proliferation, whereas it inhibited tumoral osteoblasts. GluVO, the complex with glucose, was also more toxic for tumoral than for nontransformed cells. These 2 vanadium derivatives are good potential antitumoral drugs. All the results suggest that the biological effects of vanadium compounds are a complex phenomenon influenced by the complexation, the dose, and the nature of the ligands and the cells.     

Synthesis of a new vanadyl(IV) complex with trehalose (TreVO): insulin-mimetic activities in osteoblast-like cells in culture

Vanadium compounds show interesting biological and pharmacological properties. Some of them display insulin-mimetic effects and others produce anti-tumor actions. The bioactivity of vanadium is present in inorganic species like the vanadyl(IV) cation or vanadate(V) anion. Nevertheless, the development of new vanadium derivatives with organic ligands which improve the beneficial actions and decrease the toxic effects is of great interest. On the other hand, the mechanisms involved in vanadium bioactivity are still poorly understood. A new vanadium complex of the vanadyl(IV) cation with the disaccharide trehalose (TreVO), Na(6)[VO(Tre)(2)].4H(2)O, here reported, shows interesting insulin-mimetic properties in two osteoblast cell lines, a normal one (MC3T3E1) and a tumoral one (UMR106). The complex affected the proliferation of both cell lines in a different manner. On tumoral cells, TreVO caused a weak stimulation of growth at 5 microM but it inhibited cell proliferation in a dose-response manner between 50 and 100 microM. TreVO significantly inhibited UMR106 differentiation (15-25% of basal) in the range 5-100 microM. On normal osteoblasts, TreVO behaved as a mitogen at 5-25 microM. Different inhibitors of the MAPK pathway blocked this effect. At higher concentrations (75-100 microM), the complex was a weak inhibitor of the MC3T3E1 proliferation. Besides, TreVO enhanced glucose consumption by a mechanism independent of the PI3-kinase activation. In both cell lines, TreVO stimulated the ERK phosphorylation in a dose- and time-dependent manner. Different inhibitors (PD98059, wortmannin, vitamins C and E) partially decreased this effect, which was totally inhibited by their combination. These results suggest that TreVO could be a potential candidate for therapeutic treatments.     

Antioxidant effects of the VO(IV) hesperidin complex and its role in cancer chemoprevention

Vanadium compounds are known for a variety of pharmacological properties. Many of them display antitumoral and osteogenic effects in several cell lines. Free radicals induce the development of tumoral processes. Natural polyphenols such as flavonoids have antioxidant properties since they scavenge different free radicals. For these reasons it is interesting to investigate the effects of a new complex generated between the vanadyl(IV) cation and the flavonoid hesperidin. The complex has been synthesized and characterized by physicochemical methods. Spectroscopic analysis revealed a 1:1 stoichiometry of ligand:VO and coordination by deprotonated cis-hydroxyl groups to the disaccharide moiety of the ligand. The complex improves the superoxide dismutase (SOD)-like activity of the ligand, but the scavenging of other radicals tested does not change upon complexation. When tested on two tumoral cell lines in culture (one of them derived from a rat osteosarcoma UMR106 and the other from human colon adenocarcinoma Caco-2), the complex enhanced the antiproliferative effects of the free ligand, and this effect correlated with the morphological alterations toward apoptosis. Also, on the osteoblastic cell line the complex stimulated cell proliferation and collagen type I production at low concentrations. At higher doses the complex behaved as a cytotoxic compound for the osteoblasts.     

Synthesis of vanadium(IV,V) hydroxamic acid complexes and in vivo assessment of their insulin-like activity

We synthesized vanadyl (oxidation state +IV) and vanadate (oxidation state +V) complexes with the same hydroxamic acid derivative ligand, and assessed their glucose-lowering activities in relation to the vanadium biodistribution behavior in streptozotocin-induced diabetic mice. When the mice received an intraperitoneal injection of the complexes, the vanadate complex more effectively lowered the elevated glucose levels compared with the vanadyl one. The glucose-lowering effect of the vanadate complex was linearly related to its dose within the range from 2.5 to 7.5 mg V/kg. In addition, pretreatment of the vanadate complex induced a larger insulin-enhancing effect than the vanadyl complex. Both complexes were more effective than the corresponding inorganic vanadium compounds. The vanadyl and vanadate complexes, but not the inorganic vanadium compounds, resulted in almost the same organ vanadium distribution. Consequently, the observed differences in the insulin-like activity between the complexes would reflect the potency of the two compounds in the +IV and +V oxidation states in the subcellular region.     

Anticancer activity of oxovanadium compounds

Cytotoxic and antitumor activities of the biligand vanadyl derivative of L-malic acid, (bis-(L-malato)oxovanadium(IV) (VO(mal)₂), the inorganic vanadium(IV) compound, vanadyl sulfate (VOSO₄), the oxovanadium monocomplex with L-malic acid (VO(mal)), and the vanadyl biscomplex with acetylacetonate (VO(acac)₂) were investigated using several tumor cell lines: mouse fibrosarcoma (L929), rat pheochromocytoma (PC12), human liver carcinoma (HepG2), mouse embryonic fibroblasts (NIH/3T3), and also normal human skin fibroblasts. The results showed that VO(mal)₂ effectively inhibited growth of cancer cell cultures without any toxic effect on normal human skin fibroblasts. The cytotoxic anticancer effect of vanadium complexes depended on concentration of the compounds studied, incubation time, types of cell cultures, and nature of ligands surrounding the central group of the complex (VO²⁺). These studies provide evidence that VO(mal)₂ may be considered as a potential anticancer agent due to its low toxicity for non-tumor cells and significant anticancer activity.     

Spectroscopic Characterization of a VO<Superscript>2+</Superscript> Complex of Oxodiacetic Acid and its Bioactivity on Osteoblast-like Cells in Culture

The oxovanadium(IV) complex of oxodiacetic acid (H₂oda) of stoichiometry [VO(oda)(H₂O)₂], which presents an unprecedented tridentate OOO coordination, was thoroughly characterized by infrared, Raman, electronic, and electron paramagnetic resonance spectroscopies. The biological activity of the complex on the cell proliferation and differentiation was tested on osteoblast-like cells (MC3T3E1 osteoblastic mouse calvaria-derived cells and UMR106 rat osteosarcoma-derived cells) in culture. The complex caused inhibition of cellular proliferation in both osteoblast-like cells in culture, but the cytotoxicity was stronger in the normal (MC3T3E1) than in the tumoral (UMR106) osteoblasts. The effect of the complex in cell differentiation was tested through the specific activity of alkaline phosphatase of the UMR106 cells because they expressed a high activity of this enzyme. What occurs with other vanadium compounds [VO(oda)(H₂O)₂] is an inhibitory agent of osteoblast differentiation.     

Osteogenic activity of vanadyl(IV)-ascorbate complex: evaluation of its mechanism of action

We have previously shown that different vanadium(IV) complexes regulate osteoblastic growth. Since vanadium compounds are accumulated in vivo in bone, they may affect bone turnover. The development of vanadium complexes with different ligands could be an alternative strategy of use in skeletal tissue engineering. In this study, we have investigated the osteogenic properties of a vanadyl(IV)-ascorbate (VOAsc) complex, as well as its possible mechanisms of action, on two osteoblastic cell lines in culture. VOAsc (2.5-25 microM) significantly stimulated osteoblastic proliferation (113-125% basal, p<0.01) in UMR106 cells, but not in the MC3T3E1 cell line. VOAsc (5-100 micrioM) dose-dependently stimulated type-I collagen production (107-156% basal) in osteoblasts. After 3 weeks of culture, 5-25 microM VOAsc increased the formation of nodules of mineralization in MC3T3E1 cells (7.7-20-fold control, p<0.001). VOAsc (50-100 microM) significantly stimulated apoptosis in both cell lines (170-230% basal, p<0.02-0.002), but did not affect reactive oxygen species production. The complex inhibited alkaline and neutral phosphatases from osteoblastic extracts with semi-maximal effect at 10 microM doses. VOAsc induced the activation and redistribution of P-ERK in a time- and dose-dependent manner. Inhibitors of the mitogen activated protein kinases (MAPK) pathway (PD98059 and UO126) partially blocked the VOAsc-enhanced osteoblastic proliferation and collagen production. In addition, wortmanin, a PI-3-K inhibitor and type-L channel blocker nifedipine also partially abrogated these effects of VOAsc on osteoblasts. Our in vitro results suggest that this vanadyl(IV)-ascorbate complex could be a useful pharmacological tool for bone tissue regeneration.     

Biochemical properties and mechanism of action of a vanadyl(IV) – aspirin complex on bone cell lines in culture

A recently synthesized vanadyl(IV) complex with aspirin[VO(aspirin)ClH₂O]₂, has been thoroughly investigated by physicochemical techniques. In order to support the proposed structure, stoichiometry and the coordination sphere of the vanadium center, some studies such as elemental analysis, electronic (diffuse reflectance) and vibrational (infrared) spectroscopies, magnetic susceptibility, as well as the thermal behavior, were carried out. The bioactivity of the vanadium complex (VOAspi) was evaluated on two osteoblast-like cell lines in culture, being its cytotoxic effects stronger than the vanadyl cation as assessed by morphological changes and lipid peroxidation. These effects may be partially explained through the induction of the expression of Erks (Extracellular signal-regulated kinases) and the inhibition of the PTPases (Phosphotyrosine phosphatases) present in the cellular extracts.     

The Effects of the Endocannabinoids Anandamide and 2-Arachidonoylglycerol on Human Osteoblast Proliferation and Differentiation

The endocannabinoid system is expressed in bone, although its role in the regulation of bone growth is controversial. Many studies have examined the effect of endocannabinoids directly on osteoclast function, but few have examined their role in human osteoblast function, which was the aim of the present study. Human osteoblasts were treated from seeding with increasing concentrations of anandamide or 2-arachidonoylglycerol for between 1 and 21 days. Cell proliferation (DNA content) and differentiation (alkaline phosphatase (ALP), collagen and osteocalcin secretion and calcium deposition) were measured. Anandamide and 2-arachidonoylglycerol significantly decreased osteoblast proliferation after 4 days, associated with a concentration-dependent increase in ALP. Inhibition of endocannabinoid degradation enzymes to increase endocannabinoid tone resulted in similar increases in ALP production. 2-arachidonoylglycerol also decreased osteocalcin secretion. After prolonged (21 day) treatment with 2-arachidonoylglycerol, there was a decrease in collagen content, but no change in calcium deposition. Anandamide did not affect collagen or osteocalcin, but reduced calcium deposition. Anandamide increased levels of phosphorylated CREB, ERK 1/2 and JNK, while 2-arachidonoylglycerol increased phosphorylated CREB and Akt. RT-PCR demonstrated the expression of CB₂ and TRPV1, but not CB₁ in HOBs. Anandamide-induced changes in HOB differentiation were CB₁ and CB₂-independent and partially reduced by TRPV1 antagonism, and reduced by inhibition of ERK 1/2 and JNK. Our results have demonstrated a clear involvement of anandamide and 2-arachidonoylglycerol in modulating the activity of human osteoblasts, with anandamide increasing early cell differentiation and 2-AG increasing early, but decreasing late osteoblast-specific markers of differentiation.     

Vanadium compounds

The direct effect of different vanadium compounds upon alkaline phosphatase (ALP) activity was investigated. Vanadate and vanadyl inhibited both the soluble and particulate ALP activity from UMR.106 cells and from bovine intestinal ALP. We have also shown the inhibition of ALP activity in the soluble fraction of osteoblasts by peroxo and hydroperoxo vanadium compounds. ALP activity in the particulate fraction was not inhibited by these species; nor was the bovine intestinal ALP. Using inhibitors of Tyr-phosphatase (PTPases), the soluble ALP was partially characterized as a PTPase. The major activity in the particulate fraction represents the bone-specific ALP-activity. This study demonstrates that different forms of vanadium are direct inhibitors of ALP activity. This effect is dependent on the enzymatic activity investigated and on the origin of the ALP.     

The Effect of Quercetin on the Osteogenesic Differentiation and Angiogenic Factor Expression of Bone Marrow-Derived Mesenchymal Stem Cells

Bone marrow-derived mesenchymal stem cells (BMSCs) are widely used in regenerative medicine in light of their ability to differentiate along the chondrogenic and osteogenic lineages. As a type of traditional Chinese medicine, quercetin has been preliminarily reported to promote osteogenic differentiation in osteoblasts. In the present study, the effects of quercetin on the proliferation, viability, cellular morphology, osteogenic differentiation and angiogenic factor secretion of rat BMSCs (rBMSCs) were examined by MTT assay, fluorescence activated cell sorter (FACS) analysis, real-time quantitative PCR (RT-PCR) analysis, alkaline phosphatase (ALP) activity and calcium deposition assays, and Enzyme-linked immunosorbent assay (ELISA). Moreover, whether mitogen-activated protein kinase (MAPK) signaling pathways were involved in these processes was also explored. The results showed that quercetin significantly enhanced the cell proliferation, osteogenic differentiation and angiogenic factor secretion of rBMSCs in a dose-dependent manner, with a concentration of 2 μM achieving the greatest stimulatory effect. Moreover, the activation of the extracellular signal-regulated protein kinases (ERK) and p38 pathways was observed in quercetin-treated rBMSCs. Furthermore, these induction effects could be repressed by either the ERK inhibitor PD98059 or the p38 inhibitor SB202190, respectively. These data indicated that quercetin could promote the proliferation, osteogenic differentiation and angiogenic factor secretion of rBMSCs in vitro, partially through the ERK and p38 signaling pathways.     

Effect of vanadium compounds on acid phosphatase activity

The direct effect of different vanadium compounds on acid phosphatase (ACP) activity was investigated. Vanadate and vanadyl but not pervanadate inhibited the wheat germ ACP activity. These vanadium derivatives did not alter the fibroblast Swiss 3T3 soluble fraction ACP activity. Using inhibitors of tyrosine phosphatases (PTPases), the wheat germ ACP was partially characterized as a PTPase. This study suggests that the inhibitory ability of different vanadium derivatives to modulate ACP activity seems to depend on the geometry around the vanadium atom more than on the oxidation state. Our results indicate a correlation between the PTPase activity and the sensitivity to vanadate and vanadyl cation.     

Vanadyl-and Vanadate-Induced Lipid Peroxidation in Mitochondria and in Phosphatidylcholine Suspensions

Vanadyl (V_(IV)) was found to induce rapidly developing lipid peroxidation in intact and sonicated mitochondria as well as in phosphatidylcholine suspension. The ability of vanadate (V_(V)) to induce lipid peroxidation was much less pronounced compared to that of vanadyl. The peroxidative action of vanadate on phosphatidylcholine much increased in the presence of NADH and ascorbate. Preincubation of vanadate with glucose had the same effect.

Vanadyl-induced lipid peroxidation was not essentially influenced by SOD, catalase and ethanol but was completely inhibited by butylated hydroxytoluene.

All these effects of vanadyl and vanadate are thought to participate in the insulin-like and other biological actions of vanadium.     

Model complexes for amavadine,a vanadium natural product

Amavadine is a vanadium natural product from the mushroom Amanita muscaria. Earlier reports have characterized the compound as a vanadyl (VO²⁺) complex with two N-hydroxy-αα-iminodipropionic acid ligands, but no hypothesis as to its function has yet been put forward. We report here the synthesis, isolation, and properties of bis(iminodiacetato)oxovanadium(IV) and bis(αα-iminodipropionato)oxovanadium(IV). The complex bis(ββ-iminodipropionato)oxovanadium(IV) has been prepared in solution. These complexes serve as models for Amavadine. The structures of the models are analogous to that of Amavadine, with two bidentate, singly charged ligands bonding through one oxygen and one nitrogen atom. The visible spectra suggest the possibility of 1:1 complexes in solution in addition to the 2:1 ligand to metal complexes. Preliminary electrochemical data suggest reversible V(IV) ? V(III) couples.     

Increased potency of vanadium using organic ligands

Thein vivo glucose lowering effect of orally administered inorganic vanadium compounds in diabetes was first reported in our laboratory in 1985. While both vanadate and vanadyl forms of vanadium are orally active, they are still not well absorbed. We have synthesized several organic vanadium compounds and one compound, bis(maltolato)oxovanadium(IV) or BMOV, has been extensively investigated. BMOV proved effective in lowering plasma glucose and lipids in STZ-diabetic rats when administered in drinking water over a 25 week period. The maintenance dose (0.18 mmol/kg/day) was approximately 50% of that required for vanadyl sulfate (VS). Secondary complications of diabetes were prevented by BMOV and no marked toxicity was noted. Oral gavage of STZ-diabetic rats with BMOV also reduced blood glucose levels. The ED₅₀ for BMOV was 0.5 mmol/kg, while for VS the estimated ED₅₀ was 0.9 mmol/kg. BMOV was also effective by the intraperitoneal route in STZ-diabetic rats. The ED₅₀ was 0.08 mmol/kg compared to 0.22 mmol/kg for VS. Some animals treated p.o. or i.p. remained euglycemic for up to 14 weeks. An i.v. infusion of BMOV of 0.05 mmol/kg over a 30 min period reduced plasma glucose levels by 50% while VS was not effective.     

Insulin-like Effects of Vanadium: Mechanisms of Action, Clinical and Basic Implications

Most or all mammalian cells contain vanadium at a concentration of 20 nM. The bulk of the vanadium in cells is probably in the reduced vanadyl (IV) form. Although this element is essential and should be present in the diet in minute quantities, no known physiological role for vanadium has been found thus far. In the years 1975–1980 the vanadate ion was shown to act as an efficient inhibitor of Na⁺,K⁺-ATPase and of other related phosphohydrolases as well. In 1980 it was observed that vanadate and vanadyl, when added to intact rat adipocytes, mimic the biological actions of insulin in stimulating hexose uptake and glucose oxidation. This initiated a long, currently active, field of research among basic scientists and diabetologists. Several of the aspects studied are reviewed here.     

Insulin-like Effects of Vanadium: Mechanisms of Action,Clinical and Basic Implications

Summary Most or all mammalian cells contain vanadium at a concentration of 20 nM. The bulk of the vanadium in cells is probably in the reduced vanadyl (IV) form. Although this element is essential and should be present in the diet in minute quatities, no known physiological role for vanadium has been found thus far. In the years 1975–1980 the vanadate ion was shown to act as an efficient inhibitor of Na⁺, K⁺-ATPase and of other related phosphohydrolases as well. In 1980 it was observed that vanadate and vanadyl, when added to intact rat adipocytes, mimic the biological actions of insulin in stimulating hexose uptake and glucose oxidation. This initiated a long, currently active, field of research among basic scientists and diabetologists. Several of the aspects studied are reviewed here.     

The disparity between effects of vanadate (V) and vanadyl (IV) ions on (Na+-K+)-ATPase and K+-phosphatase in skeletal muscle

On crude membrane fractions of skeletal musccle, vanadyl (IV) and vanadate (V) compounds inhibited the membrane (Na⁺K⁺)-ATPase and neutral (K⁺-)p-nitrophenylphosphatase equally with K_i 4×10^?8 mol.1^?1. Only vanadate (V) inhibited significantly the muscle (Na⁺K⁺)ATPase with K_i 1×10^?6 mol.1^?1, whereas vanadyl (IV) ions were almost without effect. Extracellular application of both forms of vanadium failed to inhibit the electrogenic (Na⁺K⁺) pump in intact mouse diaphragm fibres.     

Preparation and characterization of vanadyl complexes with bidentate maltol-type ligands; in vivo comparisons of anti-diabetic therapeutic potential

A series of 2-alkyl-3-hydroxy-4-pyrone oxovanadium(IV) compounds has been synthesized, characterized, and tested for bioactivity as potential insulin-enhancing agents. The vanadyl complexes, bis(maltolato)oxovanadium(IV), BMOV, bis(ethylmaltolato)oxovanadium(IV), BEOV, and bis(isopropylmaltolato)oxovanadium(IV), BIOV, were compared against vanadyl sulfate for glucose-lowering ability, when administered i.p. to STZ-diabetic rats, at a one-time dose of 0.1 mmol kg(-1)body weight. Blood levels of vanadium were determined at regular intervals, to 72 h, following i.p. injection. All complexes tested exceeded vanadyl sulfate in glucose-lowering ability; this effect was not correlated, however, with blood vanadium levels. Analysis of the pharmacokinetics of the disappearance of [ethyl-1-(14)C]BEOV after an oral gavage dose (50 mg kg(-1), 0.144 mmol kg(-1), in a 10 mL kg(-1) volume of 1% CMC solution) indicated clearly that metal ion-ligand dissociation took place relatively soon after oral ingestion of the complex. Half-lives of fast phase uptake and slow phase disappearance for (14)C and V were calculated from a two-compartment model for whole blood, plasma, liver, kidney, bone, small intestine, and lung, ranging from 17 min ( t(1/2)alpha for (14)C, liver) to 30 days ( t(1/2)beta for V, bone). Curves of disappearance of plasma and whole blood (14)C and V diverged dramatically within the first hour after administration of the vanadium complex.