Vanadium Bioactivity on Cells in Culture - ACS Symposium Series

Chapter 21

Vanadium Bioactivity on Cells in Culture 1,2

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Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: December 10, 1998 | doi: 10.1021/bk-1998-0711.ch021

S. B. Etcheverry and A. M . Cortizo 1

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Cátedra de Bioquímica Patológica, CEQUINOR, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 115, 1900 La Plata, Argentina

Vanadium compounds show insulin-mimetic and growth factor-like properties in different biological systems. This issue has renewed the scientific interest in the synthesis and characterization of new vanadium derivatives with potential therapeutic applications. Vanadium (V) compunds like vanadate, V-oxal, V-cit, BMV (a maltol complex), as well as pervanadate and V-NTA (a peroxovanadium derivative) induce metabolic and proliferative events on two osteoblast-cell lines (UMR106 and MC3T3E1) in culture. Vanadium (IV) compounds like vanadyl, V-tar and BMOV behave similarly. The potency of the biological effect depends on the oxidation state of vanadium, the coordination sphere and the stability of the vanadium compounds under physiological conditions. Vanadium derivatives at higher tested doses caused morphological transformation in the MC3T3E1 cells with a variable potency. The transforming effect seems to correlate with the level of cellular tyrosine phosphorylation induced by the vanadium derivatives under culture conditions. It has been known for several years, that vanadium and related compounds exert insulin-like effects on different cellular types. Comprehensive studies have been carried out on tissues and cells which are typical targets of insulin action. Among the observed actions, vanadium compounds show antilipolytic effects on adipocytes, and stimulate glucose uptake and glucose oxidation in skeletal muscle and adipocytes. Besides glycogen synthesis in the liver is stimulated by vanadium derivatives. Vanadate also increases calcium influx, inhibits Ca,Mg-ATPase in plasmatic membranes and enhances potassium uptake in intact cells (1-3). Vanadium compounds, like growth factors, act on cellular proliferation and differentiation, and they also induce protooncogene expression (2,4). However, other studies have shown that vanadium inhibits cell growth and causes cytotoxicity (5,6). This effect seems to depend on the proliferative rate of the cells in culture (7). Thus, it has been shown that in cultures of high cellular density, with low level of

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©1998 American Chemical Society In Vanadium Compounds; Tracey, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


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proliferation, vanadium does not affect the mitogenicity. On the contrary, cells grown at low cellular density and with high proliferative rate are inhibited by micromolar concentrations of vanadium. For instance, the rapidly proliferative chondrocytes (8) and Syrian hamster's embryonic cells (9) are highly sensitive to vanadium cytotoxic effects. In addition, it has been reported that vanadium compounds induced morphological transformations in different cell types (10). In the presence of vanadium derivatives, cells become shrinkage and show a condensed chromatin, with few connections among them. It has been suggested that morphological changes are associated with an increase of tyrosine phosphorylation of numerous intracellular proteins (2). The mechanism through which vanadium compounds produce growth factor like effects are not completely known. Most of the growth factors regulate the cellular levels of phosphotyrosine proteins via specific cellular receptors. This effect depends on the balance between the activities of proteintyrosine kinases (PTKs) and phosphotyrosine phosphatases (PTPases). It has been demonstrated that vanadium compounds inhibit PTPases (77). Concerning animals and human beings, vanadium compounds enter the organism primarily through the respiratory tract. Vanadium is rapidly distributed on tissues and finally is mainly retained on bones (72). On the other hand, it has been previously reported that vanadium deficiency causes growth inhibition and skeletal deformations (13). Numerous studies have shown that vanadium produces significant biological effects on skeletal tissues (74-7 7).Thus, the effects of vanadium derivatives in bone-related cells are of great interest. Glucose consumption induced by vanadium compounds on bone-related cells One of the first studied insulin-like effects of vanadium on different cells in culture was the enhancement of glucose uptake and metabolism (7). In NIH3T3 mouse fibroblasts, vanadate induced an increase in GLUT-1 (glucose transporter) mRNA, in its protein expression and in the 2-deoxiglucose uptake. This effect was dosedependent and required the presence of serum in the culture (18). In our laboratory, we have observed that different vanadium compounds (vanadate, complexes with oxalate, citrate and tartrate, and V-NTA, a peroxovanadium complex) increased the glucose consumption in the osteoblast-like cells UMR106 and MC3T3E1 after a 24-hour of incubation (79). V - N T A (25 μΜ) was the strongest tested inductor of glucose consumption in UMR106 osteoblast-like cells (250 % over basal). This observation was in agreement with previous results obtained in adipocytes (16,20,21), showing that the peroxovanadium derivatives are more potent enhancers of glucose metabolism than vanadate. The insulin-induced glucose consumption in UMR106 cells is a dosedependent function. After 24-hour incubation, the maximum effect was 160 % over basal. Thus, like insulin, vanadium compounds stimulate glucose metabolism in osteoblasts-like cells.

In Vanadium Compounds; Tracey, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

272 Mitogenic effects of vanadium compounds on bone-related cells Vanadium in the cells regulates several early events like protein phosphorylation, glucose transport, ATPases inhibition, glucose and lipid metabolism. In addition, vanadium also acts over the proliferation and differentiation of different cells in culture (10). The induction of cell growth by vanadium is similar to that produced by growth factors. The observation of this mitogenic effect requires 24-48 hs and it takes place in a narrow range of concentrations. This latter feature contrasts with the action of growth factors which occurs in a wide range of concentrations (10" - 10" M) and in a dose-response manner. In particular, using different models of bone-related cells in culture, it has been previously demonstrated the direct effect of vanadate on D N A synthesis and cell replication (14-17). We have also addressed the issue on behalf of the fate of vanadium mitogenicity by using several vanadium compounds in fibroblasts and osteoblast-like cells in culture (19,22-24). We have chosen a series of vanadium compounds for previous biological studies characterized with physicochemical properties. In our models, the proliferative effects of the vanadium derivatives were biphasic, with a maximum between 10-25 μΜ, while high doses caused cytotoxic effects. We have tested different vanadium (V) and vanadium (IV) derivatives. Among the first ones there are: vanadate, vanadium (V) complexes with citrate, oxalate and maltol (bis(maltolato dioxo vanadate(V)) (BMV)). We have also tested two peroxovanadium (V) compounds. In addition to oxovanadium (IV) cation (vanadyl), complexes of vanadium (IV) with tartrate and maltol (bis(maltolato) oxovanadium (IV)) (BMOV)) were also studied. The potency of these vanadium compounds was similar for vanadium (V) and vanadium (IV) derivatives with the exception of peroxovanadium compounds that were less effective and more cytotoxic for all the cellular types studied. The sensitivity of the cells towards the vanadium derivatives seems to depend on the stage of cellular development and also on the cellular density in the cultures. Thus, comparing 70% confluent cultures of Swiss 3T3 fibroblasts, MC3T3E1 osteoblast-like cells in the proliferative phase and the more differentiated osteoblast line UMR106, the latter showed less sensitivity to the cytotoxic effect of vanadate and vanadyl. On the other hand, it has also been suggested that the cytotoxicity depends on the proliferative rate of the cell type. The cytotoxic effects of vanadium complexes seem to be dependent on several factors: the vanadium concentration in the culture media, the oxidation state of the vanadium atom, the coordination sphere, and likely on their ability to inhibit protein tyrosine phosphatases (PTP-ases). In short, the cytotoxicity is a complex phenomenon under the influence of factors inherent to the cells and physicochemical properties of the vanadium compounds.


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Morphological transformations. The mitogenic and cytotoxic effects produced by vanadium compounds were accompanied by morphological changes (19,22,24). Swiss 3T3 fibroblasts and



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MC3T3E1 osteoblast-like cells in the proliferative stages showed a shape change from polygonal to fusiform, a retraction with citoplasm condensation and a loss of lamellar processes. The magnitude of the morphological transformations correlates with the potency of vanadium compounds to induce cytotoxic effects. The vanadium compounds under study in our laboratory can be divided in three groups according with their decreasing potency as morphological transformer agents. The peroxovanadium compounds, pervanadate and V-NTA, formed the more potent group because they induce the most striking and fastest cellular changes. Vanadate, V-cit, V-oxal and B M O V are in the second group which causes intermediate morphological transformations and, finally, the third group is constituted by vanadyl cation, V-tar (a vanadium (IV) complex) and B M V . From our morphological studies, it can be suggested that in general, peroxovanadium compounds are more potent than vanadium (V) derivatives and these are more potent than vanadium (IV) compounds, with the exception of the maltol derivatives which behave contrarily taking into account the oxidation state of vanadium.

Vanadium derivatives inhibit osteoblast-like cell differentiation Studies on vanadium bioactivity have lead to address another growth factor mimetic property of these compounds, that is the ability to regulate cellular differentiation. Previous reports have demonstrated that low vanadate doses induce the synthesis of collagen and extracellular matrix proteins (14-16). On the other hand, the action of vanadate on the alkaline phosphatase activity of calvaria cells in culture have shown ambiguous results (14,16). In recent studies we have shown that vanadium compounds regulate phenotype characteristics of UMR106 osteoblast-like cells as assessed by the levels of alkaline phosphatase activity (19,23,24). Vanadate and vanadium (V) derivatives (vanadium complexes with oxalate and citrate, V N T A (a peroxo vanadium compound), and also the B M O V were inhibitors of A L P activity at proliferative doses (5-25 μΜ). On the contrary, vanadium (IV) derivatives (vanadyl sulfate and V-tar) as well as B M V , have shown no significant effect on the cellular A L P activity or they behave as weak inhibitors in these systems. Our results are in agreement with the early Canalis' paper (14) who showed the inhibition of A L P activity in calvaria rat cells by 10 μΜ vanadate after a 96-hour incubation. These studies suggest that the coordination of vanadium, its oxidation state and the stability of vanadium compounds under physiological conditions play a role in the cell differentiation process.

Direct effect of vanadium compounds on osteoblast-derived alkaline phosphatase Not only us, but also other investigators have previously demonstrated the vanadiuminhibition of acid, neutral and alkaline phosphatases using cell-free in vitro assays (2,23,25-27). In general, all vanadium compounds tested inhibit phosphatases activity



274 with E D 5 0 in the order of micromolar concentrations. Specifically, alkaline phosphatase activity associated with particulate- and soluble-UMR106 cells are inhibited at 20 % and 40 % respectively, by 100 μΜ vanadate or vanadyl (26). Since we evaluate the osteoblast differentiation by measuring the A L P activity, we have also investigated the possibility of a direct inhibition of vanadium compounds on cell associated-ALP. For these studies, we determined the A L P by histochemistry after an overnight incubation with or without 100 μΜ vanadium compounds in serum-free medium. Cells were washed with PBS, fixed and stained with naphtyl phosphate and fast blue for 15 min at room temperature (24). Preincubation of UMR106 cells with or without vanadate or vanadyl did not affect the A L P staining associated with the osteoblast-like cells. However, when the same concentration of vanadate or vanadyl was added to the A L P histochemical assay, the staining almost disappeared. These results suggest that vanadium compounds exert a direct effect on the A L P activity but this inhibition is blunted by washing the cell monolayer. These experiments also suggest that the previously described studies on inhibition of cell differentiation by vanadium compounds are not due to the direct effect of vanadium but rather on the A L P expression on UMR106 cells. Biological mechanism of vanadium compounds The precise mechanisms by which vanadium compounds produced their metabolic, mitogenic and transforming effects still remain unestablished. It is well known that insulin and growth factors promote cell growth by the interaction with specific cellular surface receptors. This interaction leads to the autophosphorylation of the receptors and consequently to the phosphorylation cascade of several intracellular proteins specifically on tyrosine residues (28). Most of the evidence suggests that vanadium compounds can act on some steps of these pathways (29). Thus, different intracellular proteins become phosphorylated upon vanadium stimulation. It is assumed that vanadium-induced protein tyrosine phosphorylation is mediated mainly by the inhibition of different protein tyrosine phosphatases. In the MC3T3E1 osteoblast-like cells in culture, we have investigated the effects of different vanadium compounds on the protein tyrosine phosphorylation levels in order to understand the possible mechanism of vanadium action (30). We have also attempted to correlate these patterns with the vanadium-induced growth and cell transformation. Vanadate, vanadyl and B M O V appear to be more potent than B M V in stimulating protein phosphorylation in thyrosine residues. This effect was more prominent at low doses than at high doses. At low doses (10 μΜ), B M V showed a phosphorylation pattern similar to that of insulin, while V , V O and B M O V induced a strong phosphorylation of cell proteins. In our osteoblast system, a 90-95 kDa band presumably corresponding to the insulin-receptor β-subunit was not observed. The present findings suggest that the vanadium-induced growth regulation and transformation in MC3T3E1 osteoblast-like cells are strongly associated with the ability of these agents to increase the phorphotyrosine protein levels.


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Conclusions


Studies concerning the biological effects of vanadium compounds on bone related cells lead to the conclusion that they play a role on hard tissues growth and development. Our results show that vanadium compounds with weaker effect on the inhibition of osteoblast differentiation, as evaluated by alkaline phosphatase activity, are also the weakest transformer agents. These effects correlate with the lesser extension of tyrosine protein phosphorylation induced by these compounds. The model system used in these studies let us evaluate the mechanism by which vanadium compounds could be accumulated and metabolized in the bone. Aknowledgments SBE and AMC are indebted to VC Sâlice, DA Barrio, MD Braziunas and CM Vescina, all of them members of their research team. The authors wish to thank Mrs. MC Bernai for the language revision. SBE is a member of the Carrera del Investigador Cientifico (CONICET, Argentina). AMC is a member of the Carrera del Investigador Cientifico (CICPBA, Argentina). The laboratory work reported herein has been supported by the Facultad de Ciencias Exactas (UNLP), the Universidad Nacional de La Plata (UNLP) (Argentina), and the Third World Academy of Sciences (Italy). Literature Cited 1. Shechter, Y.; Shisheva, A. Endeavour 1993,17,27-31. 2. Stern, Α.; Yin, X.; Tsang, S-S.; Davison, Α.; Moon, J. Biochem. Cell Biol. 1993, 71, 103-112. 3. Sekar, N.; Li, J.; Shecheter, Y. Crit. Rev. Biochem. Mol. Biol. 1996, 31, 339-359. 4. Wang, H.; Scott, R.E. Mol. Cell. Biochem. 1995, 153, 59-67. 5. Sabbioni, E.; Pozzi, G.; Pintar, Α.; Casella, L.; Garattini, S. Carcinogenesis 1991, 12, 47-52. 6. Sabbioni, E.; Pozzi, G.; Devos, S.; Pintar, Α.; Casella, L.; Fishbach, M. Carcinogenesis 1993, 14, 2565-2568. 7. Cruz, T.F.; Morgan, Α.; Min, W. Mol. Cell. Biochem. 1995, 153, 161-166. 8. Conquer, J.A..; Grima, D.T.; Cruz, T.F. Ann. New York Acad. Sci. 1994, 732, 447-450. 9. Afshari, C.A.; Kodama, S.; Bivins, H.M.; Willard, T.B.; Fujiki, H.; Barrett, J.C. Cancer Res. 1993, 53, 1777-1782. 10. Etcheverry, S.B.; Cortizo, A.M. In Vanadium in the enviroment, Part I: Chemistry and biochemistry; NriaguJ.O., Ed.; John Wiley & Sons, Inc., New York, 1998,Vol 1, pp 359-394. 11. Gresser, M.J.; Tracey, A.S.; Stankiewicz, P.J. Adv. Prot. Phosphatases 1987, 4, 35-57. 12. Nielsen, F.H. In Vanadium and its role in life; Siegel, H., Siegel A. Eds. Marcell Dekker, New York, 1995, vol 31, pp 543-573.


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13. Anke, M.; Groppel, B.; Gruhn, K.; Langer, M.; Arnhold, W. In6 International Trace Element Symposium, Molybdenum, Vanadium Anke, M., Baumann, W., Bräunlich, H., Brücker, C., Groppel, B., Grün, M. Eds., Karl-Marx, Universität, Leipzig, 1989, Vol 1, pp 17-27. 14. Canalis, E. Endocrinology 1985, 116, 855-862. 15. Kato, Y.; Iwamoto, M.; Koile, T.; Suzuki, F. J. CellBiol.1987, 104, 311-319. 16. Lau, K.H.W.; Tanimoto, H.; Baylink, D.J. Endocrinology 1988, 123, 2858-2867. 17. Davidai, G.; Lee, Α.; Schuartz, Y.; Hazum, E. Am. J. Physiol. 1992, 263, E205E209. 18. Mountjoy, K.G.; Flier, J.S. Endocrinology 1990, 127, 2025-2034. 19. Etcheverry, S.B.; Crans, D.C.; Keramidas, A.D.; Cortizo, A.M. Arch Biochem. Biophys. 1997, 338, 7-14. 20. Fantus, I.G.; Kadota, S.; Deragon, G.; Foster, B.; Posner, B.I. Biochemistry 1989, 28, 8864-8871. 21. Shisheva, Α.; Shechter, Y. Endocrinology 1993, 133, 1562-1568. 22. Cortizo, A.M.; Sálice V.C.; Vescina, C.M.; Etcheverry, S.B. Biometals 1997, 10, 127-133. 23. Cortizo, A.M.; Etcheverry, S.B. Mol. Cell Biochem. 1995, 145, 97-102. 24. Barrio, D.A.; Braziunas, M.D.; Etcheverry, S.B.; Cortizo, A.M. J. Trace Elements Med. Biol. 1997, 11, 110-115. 25. Crans, D.C.; Bunch, R.L.; Theisen, L.A. J. Am. Chem. Soc. 1989, 111, 75977607. 26. Cortizo, A.M., Sálice, V.C., Etcheverry, S.B. Biol. Trace Elemen. Res. 1994, 41, 331-339. 27. Vescina, C.M.; Sálice, V.C.; Cortizo, A.M.; Etcheverry, S.B. Biol. Trace Elemen. Res. 1996, 53, 185-191. 28. Cheatham, B.; Kahn, C.R. Endocrine Rev. 1995, 16, 117-142. 29. Goldfine, A.B.; Simonson, D.C.; Folli, F.; Patto, M.-E.; Kahn, C.R. Mol. Cell. Biochem. 1995, 153, 217-231. 30. Sálice, V.C., Cortizo, A.M., Gómez Dumm, C.L., Etcheverry, S.B. Submitted.


Vanadium Bioactivity on Cells in Culture - ACS Symposium Series

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