Cytotoxicity of 1, 2, 3, 4 and 5 against PC-3, Z-138 and JURKAT cancer cell lines and HMLE, MCF 10A, 1BR3G and CCD-18Co non malignant cells.

<p>^<i>a</i> The IC_<b>50</b> values were determined by the MTT assay after 48 h of treatment. Data represents the mean ± SD of at least three independent experiments made in triplicates.</p><p>^{<b><i>b</i></b>} Percentage of hemolysis at 100 μM.</p><p>Cytotoxicity of 1, 2, 3, 4 and 5 against PC-3, Z-138 and JURKAT cancer cell lines and HMLE, MCF 10A, 1BR3G and CCD-18Co non malignant cells.</p

Mode of action of the amine-pyridine ligands.

<p>The cytotoxic activity of amine-pyridine ligands involves different inter-dependent processes: intracellular Fe(II) chelation, generation of ROS, DNA fragmentation through oxidative mechanisms, induction of cell cycle arrest and apoptosis.</p

Induction of caspase activity.

<p>(A) The indicated cell lines were treated for 48 h with compounds <b>1</b>, <b>2</b> and <b>5</b> (10 μmol/L) and caspase 3/7 activity was measured as indicated in Materials and Methods. Bar charts show the fold increase in caspase activity relative to untreated (control) and represent the mean±SD from three independent experiments performed in triplicate. (B) MCF-7 and CAPAN-1 cells were treated for 48 h with the ligands (10 μM) in the absence (-) or presence (+) of the pan-caspase inhibitor QVD-Oph (20 μM) and cell viability was measured with the MTT assay. The data shows the percentage of viable cells relative to untreated cells (control). Data represents the mean ± SD of 3 independent experiments performed in triplicate. The differences between absence and presence of QVD-OPh treatment were statistically significant at * p<0.05, **p<0.01 and ***p<0.001.</p

Effects of iron on 1, 2 and 5 activity.

<p>(A) Effect of 2 h FeCl₂ pre-incubation (FeCl₂ p) (dotted line) or co-incubation (FeCl₂ c) (dashed line) at 1, 10 or 100 μmol/L on the cytotoxicity of <b>2</b> (5 μmol/L) in MCF-7 and CAPAN-1 cells. FeCl₂ alone was included as a control (solid line). Cell viability was determined after 48 h of treatment by the MTT assay. (B) Effect of FeCl₂ pre-incubation or co-incubation (100 μmol/L) on the cytotoxicity of <b>2</b> (5 μmol/L), <b>1</b> and <b>5</b> (10 μmol/L). Cells treated with the ligands alone were included as a reference (control). (C) ROS induction in CAPAN-1 cells after 24 h treatment with <b>2</b> (10 μmol/L) alone, together with FeCl₂ or after FeCl₂ pre-incubation (at 100 μmol/L). Results are presented as a percentage of untreated cells and represent mean±SD of 3 independent experiments. *p < 0.05 versus control cells.</p

Effect of ligands on cell cycle distribution.

<p>MCF-7 cells (left) and LN229 cells (right) were left untreated (top) or treated with the indicated ligands (10 μmol/L) for 24 and 48 h. Cell cycle profiles were obtained by flow cytometry of propidium iodide-stained cells. The quantified percentage of cells in each cell cycle phase is indicated.</p

Structures of the iron complexes and the corresponding iron-free organic compounds.

<p>Structures of the iron complexes and the corresponding iron-free organic compounds.</p

Cell viability assay.

<p>(A) Cytotoxic activity of compounds <b>1</b>, <b>2</b>, and <b>5</b> against cancer cells. The indicated cell lines were treated for 48 h with ligands (10 μmol/L) and cell viability was measured with the MTT assay. Data represents the percentage of viable cells relative to untreated cells (control). (B) Cytotoxicity of doxorubicin and compounds <b>1</b>, <b>2</b> and <b>5</b> against CS-like cells. CS-like HMLER-shEcad cells and non-CS-like HMLER isogenic parental cells were treated for 48 h with graded concentrations of doxorubicin, <b>1</b>, <b>2</b> and <b>5</b>. Cell viability was measured with the MTT assay. For each treatment, the percentage of viable cells relative to untreated cells is indicated. Data represents the mean±SD of 3 independent experiments performed in triplicate.</p

Cytotoxicity of the iron complexes and the corresponding ligands against MCF-7 and CAPAN-1 cells.

<p>^<i>a</i>The IC₅₀ values were determined by the MTT assay after 48 h of compound exposure. Data represents the mean ± SD of at least three independent experiments made in triplicates.</p><p>Cytotoxicity of the iron complexes and the corresponding ligands against MCF-7 and CAPAN-1 cells.</p

Relative proportions of different forms of plasmid DNA after 1, 1-Fe, 2, 2-Fe, 5 and 5-Fe treatments.

<p>* I: supercoiled form, II: open form.</p><p>Relative proportions of different forms of plasmid DNA after 1, 1-Fe, 2, 2-Fe, 5 and 5-Fe treatments.</p

Effects of NAC on compound 1, 2 and 5 activity.

<p>CAPAN-1 cells were incubated with 10 μmol/L of <b>1</b>, <b>2</b> or <b>5</b> for 24 h with or without NAC (5 mmol/L). (A) Changes in the ROS levels were determined with H₂DCFDA staining. (B) Changes in cell viability were determined by MTT assay. The results are presented as a percentage of untreated cells and represent mean±SD of 3 independent experiments. *p < 0.05 versus control cells. ^#p < 0.05 versus cells without NAC treatment.</p