Figure 1.
Line drawings of the chemical structures of the iron chelators, SIH, HAPI and HPPI, and their novel analogs.
Table 1.
Chromatographic conditions used for the determination of the stability of the new chelators in rabbit plasma.
Table 2.
Molecular weights (MW) and calculated n-octanol/water coefficients (log Pcalc) of the studied analogs.
Figure 2.
Stabilities of SIH and its novel analogs in rabbit plasma.
SIH (A), redSIH (B), redHAPI (C), redHPPI (D), BHAPI (E), BHPPI (F), 2API (G), 7HII (H), H16 (I), H17 (J), H18 (K) and H28 (L) were incubated at 37°C in rabbit plasma and their concentrations were analyzed using HPLC every 60 min until t = 600 min. Results are expressed as a percentage of the concentration at t = 0 min (100 µM). Results are Mean ±SD (n = 3 experiments). Statistical significance (ANOVA): * p<0.05, ** p<0.01, *** p<0.001 compared to the concentration at t = 0 min (100%).
Figure 3.
Iron chelation properties of the novel analogs in solution (A) and in MCF-7 cells (B).
(A) The chelation dynamics of the new agents in solution were observed for 360 s using the calcein assay, and the agent was applied at t = 100 s. The fluorescence intensity of free calcein at t = 360 s was expressed as a percentage of that observed using the reference iron chelator, SIH. (B) The ability of the analogs to chelate “free” iron from the LIP in MCF-7 cells was measured using the calcein-AM assay. The fluorescence intensity of free calcein at t = 600 s was expressed as a percentage of that observed in the presence of SIH. Results are Mean ±SD (n≥3 experiments). Statistical significance (ANOVA): # p<0.05, ## p<0.01, ### p<0.001 compared to the reference chelator, SIH.
Figure 4.
The effect of SIH and its analogs on 59Fe mobilization from pre-labeled MCF-7 cells (A) and on internalized 59Fe uptake from 59Fe2-transferrin (Tf) by MCF-7 cells (B).
(A) The ability of the ligands to promote 59Fe mobilization from MCF-7 cells was performed by first prelabeling the cells with 59Fe2-Tf (0.75 µM) for 3 h/37°C, followed by washing and then reincubation for 3 h/37°C with either control medium alone, or control medium containing the chelator (25 µM). (B) Inhibition of 59Fe uptake from 59Fe2-Tf by MCF-7 cells by chelators was performed by incubating cells for 3 h/37°C with 59Fe2-Tf (0.75 µM) in the presence or absence of the chelator (25 µM). Results are Mean ±SD (n≥3 experiments). Statistical significance (ANOVA): * p<0.05, ** p<0.01, *** p<0.001 compared to the control (untreated) group, and # p<0.05, ## p<0.01, ### p<0.001 compared to the reference chelator, SIH.
Figure 5.
Effects of SIH and its analogs on iron-induced oxidation of ascorbic acid in a buffered solution (pH 7.4).
Chelators were assayed at iron binding equivalents (IBE) of 0.1 (excess of Fe), 1 (iron-chelator complexes with a fully filled coordination sphere) and 3 (excess of free chelator). DFO and EDTA were used as negative and positive control chelators, respectively. The results are expressed as a percentage of the control group in the absence of chelator (100%). Results are Mean ±SD (n≥3 experiments). Statistical significance (ANOVA): * p<0.05, ** p<0.01, *** p<0.001 as compared to the control group (iron with ascorbate).
Figure 6.
Protective effects of the chelator, SIH (A), and the new analogues (B–L).
The ability of the SIH derivatives to protect H9c2 cardiomyoblast cells against oxidative injury were evaluated using a 24 h/37°C incubation of the cells with H2O2 (200 µM) and the novel analogs (0.3–1000 µM). Results are Mean ±SD (n≥4 experiments). Statistical significance (ANOVA): * p<0.05, ** p<0.01, *** p<0.001 compared to the control (untreated) group, and # p<0.05, ## p<0.01, ### p<0.001 compared to the H2O2 group.
Table 3.
Protective and cytotoxic effects of the synthesized SIH derivatives and their calculated “selectivity ratios”.
Figure 7.
Cytotoxic effects of the chelator, SIH (A), and the new analogues (B–L), using non-tumorigenic H9c2 cardiomyoblasts.
The effect of the analogs (0.3–300 µM) on the cellular viability of H9c2 cardiomyoblasts were performed using a 72 h/37°C incubation. Results are Mean ±SD (n≥4 experiments). Statistical significance (ANOVA): * p<0.05, ** p<0.01, *** p<0.001 as compared to the control (untreated) group.
Figure 8.
Cytotoxic effects of the chelator, SIH (A), and the new analogues (B–L) against MCF-7 breast cancer cells.
For the determination of their cytotoxic activity, MCF-7 breast adenocarcinoma cells were incubated with the analogs (0.01–3000 µM) for 72 h/37°C. Results are Mean ±SD (n≥4 experiments). Statistical significance (ANOVA): * p<0.05, ** p<0.01, *** p<0.001 as compared to the control (untreated) group.