Alejandra Rodriguez, Javier Garcia, Patrick Gameza, Julia Lorenzo and Virtudes Moreno
Posters-Accepted Abstracts: Biol Syst Open Access
Most current research efforts with respect to the quest for novel metal-based drugs are devoted to investigating their interaction with the DNA double helix considering the accepted mechanism of action for the well-established anti cancer drug cisplatin. However, this approach neglects the fact that other cellular components may be targeted by metal complexes. In the present study, fibronectin and tubulin, two proteins involved in fundamental cellular processes that are cell division and cell migration have been chosen as (cytotoxic) targets for copper (II) and a ruthenium (II) compounds. The potential interaction of a series of metal-based molecules with these two proteins was first assessed by circular dichroism (CD) and atomic-force microscopy (AFM). MTT assays were subsequently used to determine the cell-growth inhibitory activities (IC50) of the compounds in HeLa and HL60 cell lines. Immunofluorescence assays were then carried out with the two most cytotoxic metal complexes with HeLa cells using anti-���±, anti-���²-tubulin and anti-fibronectin antibodies to investigate their effect on microtubules and the extracellular matrix. The microtubule-depolymerizing agent Nocodazole was used as positive control. Cell cycle analyses by flow cytometry and annexin V-FITC+PI apoptosis assays (with cisplatin as positive control) were performed with both cell lines to better understand the mechanisms of action of the two compounds. The AFM and CD experiments clearly evidenced the interaction of the Cu (II) and Ru (II) compounds with both proteins, the most efficient being the copper molecule. IC50 values lower than those of the reference compound cisplatin were obtained for both the Ru (II) (13.01 ���¼M HeLa; 2.48 ���¼M HL60) and the Cu (II) (3.63 ���¼M HeLa; 14.50 ���¼M HL60) complexes. The immunofluorescence assays revealed a microtubule-depolymerizing behavior for the copper molecule and the formation of apoptotic nuclei with both compounds. The cell cycle tests did not show an arrest at the G2 phase which would have indicated microtubule stabilization (that prevents cell division), therefore suggesting that the cells had died by apoptosis as confirmed by the annexin assays. In summary, fibronectin and tubulin are legitimate target proteins for potential metal-based anticancer drugs such as the two compounds evaluated herein which showed apoptotic cytotoxic activities.
