A time–response analysis of G8 and G12 in relation to caspase-3 activity was performed initially. B16F10 cells (1 × 106) were treated with 50 μM G8 and G12 for periods of time ranging from 15 min to 24 h (15 min and 1, 2, 4, 6 and

24 h). Evaluation of caspase-3 activity was performed fluorimetrically by monitoring the cleavage of the Ac-DEVD-AMC caspase-3 fluorogenic substrate. A significant increase of caspase-3 activity was observed at the initial times Selleckchem Sirolimus (between 15 min and 1 h) of incubation with G8 and G12 (Fig. 3a and b). In addition, the protease activity did not change significantly when comparing the untreated control with samples incubated from 2 to 24 h with the gallates. The mitochondrial membrane potential was investigated after 15 min of incubating B16F10 cells (3 × 105) with 50 μM GA, G8 and G12 using the JC-1 fluorescent probe and 5 μM FCCP as a positive control. In this experiment, a decrease in mitochondrial

membrane potential in response to G8 and G12 incubation was observed, as evidenced by the decrease in the intensity of the red and green fluorescence ratio (Fig. 4a and b). This result suggests the involvement of the intrinsic pathway of apoptosis in the mechanism of cell death induction by the gallates. Fas death-receptor activation is associated with apoptosis initiated by the extrinsic pathway, and the Bcl-2 family members, Bcl-2 and Bax, account for anti- and proapoptotic functions, respectively. Assessments of the Fas death receptor (Fas) and the anti- and proapoptotic proteins’ (Bcl-2 and

selleck kinase inhibitor Bax) expression were performed in an incubation time of 3 h with gallates. The results indicate that G8 and G12 did not promote changes in the expression of cell death pheromone receptor Fas (Fig. 5a); however, G8 and G12 promoted a significant increase in the expression of the proapoptotic protein Bax (Fig. 5b) and a decrease in the anti-apoptotic protein Bcl-2 level (Fig. 5c). Catalase activity, lipoperoxidation and free radical generation were evaluated in B16F10 cells incubated with 50 μM G8 and G12. The effects on non-protein thiol content (GSH, GSSG and GT), as well as on the activity of gamma glutamyl cysteine synthase (GGCS), were evaluated in previous studies by this laboratory (Locatelli et al., 2009). B16F10 cells (3 × 106) were incubated for 3 h with 50 μM AG, G8 and G12 or with 1 mM H2O2 and 200 μM Trolox, which were used as positive and negative controls, respectively. Cell extracts were obtained for the evaluation of lipid peroxidation and catalase activity. In B16F10 cells treated with gallates, free radical generation was analyzed qualitatively and quantitatively using the DCFDA fluorescent probe. G8 significantly increased the lipid peroxidation and reactive species generation in relation to the control in 34% ± 1 (Fig. 6c) and 123% ± 11 (Fig. 6a and b), respectively. G12 increased reactive species generation in relation to the control in 69% ± 8 (Fig. 6a and b) but did not promote lipid peroxidation (Fig. 6c).