Biological Effects and Cytotoxicity of a complex of Vanadium(V) with salicylaldehyde semicarbazone in osteoblasts in culture

JOSEFINA RIVADENEIRA, DANIEL A. BARRIO, GABRIEL ARRAMBIDE, DINORAH GAMBINO, LILIANA BRUZZONE, SUSANA B. ETCHEVERRY

Lisboa, Portugal

Simposio; Sixth International Vanadium Symposium; 2008

Biological Effects and Cytotoxicity of a complex of Vanadium(V) with salicylaldehyde semicarbazone in osteoblasts in culture.   Josefina Rivadeneiraa,b, Daniel A. Barrioa, Gabriel Arrambidec, Dinorah Gambino c, Liliana Bruzzonea, Susana B. Etcheverrya,b*   aFacultad de Ciencias Exactas, UNLP. 47 y 115 (1900) La Plata, Argentina bCEQUINOR (CONICET-UNLP), Facultad de Ciencias Exactas, UNLP, La Plata, Argentina cFacultad de Química, Universidad de la República, Montevideo, Uruguay * Corresponding author E-mail: etcheverry@biol.unlp.edu.ar   Interaction of simple vanadium species with ligand groups bearing pharmacological activity, particularly those with antitumoral and insulin-mimetic properties, is of growing scientist interest. Vanadate mimics various insulin actions on different cell types and also in cell free systems. Semicarbazones are versatile compounds of considerable interest because of their chemistry and potentially beneficial pharmacological effects, such as antitumor, antibacterial, antiviral and antimalarial activities. The biological effects of these ligands are considered to be related to their ability to form chelates with metals. Biological activities of metal complexes differ from those of either ligands or the metal ions, and increased or decreased biological activities have been reported for several transition metal complexes. In the frame of our continuing studies devoted to the biological and potential pharmacological properties of vanadium compounds we report herein the biological actions of VVO2(salicylaldehydesemicarbazone) (V(V)-SalSem) on two osteoblast cell lines in culture (MC3T3E1 and UMR106). Cell proliferation, differentiation, morphological alterations, oxidative stress, activation of the extracellular regulated kinases (ERK) cascade and apoptosis have been investigated. V(V)-SalSem inhibited cell proliferation in a dose response manner as determined by the crystal violet bioassay, with the same potency and efficacy in both cell lines (IC50: >100 mM). The inhibition at high doses (100mM) could be partially reversed by the free radical scavengers NAC (N-acetylCysteine) and a mixture of vitamins E and C. Changes in cell proliferation correlated with morphological alterations as could be determined by ligt microscopy with Giemsa staining. Alterations began at 10 mM and increased with complex concentration. Stress fibers were also desorganized in a dose response manner being the network lost between 50-100 mM. Specific activity of alkaline phosphatase (ALP) and collagen content, two mature osteoblast phenotype markers, were inhibited in a dose response manner by the complex in UMR106 cells. In an atempt to elucidate the mechanisms of action involved in the toxicity actions of V(V)-SalSem, the oxidative stress and the activation of ERK pathway were analyzed. The determination of oxidative stress through the fluorometric measurement of free radical levels, showed that complex promoted the production of free radicals (Dihydrorhodamine oxidation to rhodamine) in a dose response manner reaching a two-fold value at 100 mM in comparison with basal conditions. This effect could be partially reversed by free radical scavengers. ERK cascade was activated by the complex as it was determined by Western blot using two specific inhibitors (PD98059) and wortmannin. These events correlated with the enhancement of apoptosis over necrosis as could be seen using AnnexinV-Propidium Iodide. In conclusion, the complex formed between vanadium(V) and salicylaldehyde semicarbazone displays cytotoxic effects on osteoblasts in culture through the production of free radicals and the activation of ERK cascade. These mechanisms triggered the apoptotic events that convey to cell death.