VO(oda): a vanadyl(IV) complex with an OOO-donor group. Bioactivity on human colon adenocarcinoma Caco-2 cell line

JOSEFINA RIVADENEIRA,CECILIA I. MUGLIA, ENRIQUE J. BARANA, LILIANA BRUZZONE, SUSANA B. ETCHEVERRY

Lisboa, Portugal

Simposio; Sixth International Vanadium Symposium; 2008

VO(oda): a vanadyl(IV) complex with an OOO-donor group. Bioactivity on human colon adenocarcinoma Caco-2 cell line.   Josefina Rivadeneiraa,b,Cecilia I. Mugliaa, Enrique J. Barana,b, 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 * Corresponding author E-mail: etcheverry@biol.unlp.edu.ar   Vanadium is a trace element widespread distributed in nature. Vanadium compounds have atracted scientific attention due to their potential therapeutic applications. The pharmacological effects of vanadium include insulin mimetic actions, osteogenic and antitumoral effects. In particular, the antitumoral actions of vanadium can be evaluated by the determination of different parameters such as cell proliferation, morphology and disruption of cellular architecture. Vanadium compounds exert their antineoplastic actions by different mechanisms (inhibition of key protein tyrosin phosphatases, changes in cell cytoskeleton proteins, and disturbance of redox equilibria in the cells that causes an increment in the oxidative stress and alterations in the ratio GSH/ GSSG). Altogether, these effects may lead to the induction of apoptosis and/or necrosis, and finally to cell death. On the other hand, strong chelating ligands are very important in aqueous media because they offer the opportunity of trapping different metal species. This process becomes particularly significant in living systems, where different metals are acquired, transported and stored mostly by low-molecular-weight compounds involving multidentate oxygen donors. Among the family of multidentate oxygen donor species, oda= oxydiacetate, O(CH2COO-)2, stands as a versatile complexing agent. It holds an OOO donor group and can complex metal ions by forming chelate rings. The synthesis of the complex VO2+-oda has been previously reported. The aim of the present study is to extend our previous observations on the bioactivity of VO2+-oda on tumoral cells, focusing the attention on its cytotoxicity effects on the human colon adenocarcinoma cell line Caco-2, trying to understand its mechanisms of action. The complex caused an inhibitory effect on cell proliferation in a dose response manner in the range of 10-100 µM (p<0.001) (Crystal violet assay). Nevertheless, this inhibitory effect was lower than the inhibition in other tumoral cell line (UMR106 rat osteosarcoma) studied in our laboratory. IC50 for the complex in Caco-2 cell line was >> 100 µM. This inhibition of Caco-2 proliferation was reversed by scavengers of free radicals as a mixture of vitamin C and E (50 µM). The reversion was complete in the range of low doses of the complex (10-25 µM) and only partial at higher concentrations (50-100 µM). Morphological studies were carried out using Giemsa staining and  light microscopy. In basal conditions, the cells displayed polygonal shape and big nuclei with numerous nucleoles. Cells present also numerous connections between each other. Upon incubation with the complex, the nuclei showed different alterations in their form and some membrane blebs could be seen. Besides, the citoplasm of the cells also showed important transformations such as numerous irregular vacuoles and lost of neighboring connections. These changes increased with the doses and correlated with a decrease in number of cells per field. Moreover, the investigation of the effects of VO2+-oda on the actin filaments, indicated a disassembly of the network as a function of complex concentration. Thus, the morphological alterations were in parallel with the results of proliferation study. In order to get a deeper insight into the mechanisms underlying the cytotoxicity of VO2+-oda, we determined its effect on the oxidative stress using two parametrs: the oxidation of dihydrorhodamine 123 to rhodamine (measured by spectrofluorometry) and the changes in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG). The complex produced a great increment in the levels of rhodamine in the range of 50-100 µM while a decrease in the GSH/GSSG ratio could be observed. Besides, the mixture of vitamins C and E, scavenger of free radicals, caused a parcial reversion of the VO2+-oda effect  in the range of complex concentration 50-100 µM. Altogether these results suggest that VO2+-oda exerts its cytotoxicity effects on human colon adenocarcinoma (Caco-2) cells through, at least in part, an increment in the oxidative stress and the alterations of actin cellular network.