Redox cycle of myoglobin.

<p>Ferric Mb reacts with hydrogen peroxide to give the long-lived oxoferryl species and a transient protein radical (tryptophanyl radical), directly formed or resulting from electron transfer from the protein to the porphyrin π cation radical (steps <i>a</i> and <i>b</i>). The long-lived oxoferryl Mb can recycle to the resting form by two electron reduction (step c). Tryptophanyl free radical could react with molecular oxygen to form a peroxyl-derived free radical (step <i>d</i>). The protein radical may lead to the Mb degradation (bleaching) as shown in step <i>e</i>.</p

Mechanism of TOP oxidation by hydrogen peroxide.

<p>Mechanism of TOP oxidation by hydrogen peroxide.</p

Production of TOP thiyl radical by Mb.

<p>The Mb-catalized one-electron oxidation of TOP is expected to generate thiyl free radical and the fate of this free radical is proposed according to reference 55.</p

Proposed mechanism of TOP participation in the redox cycling of myoglobin.

<p>In the presence of TOP, steps <i>a</i> and <i>b</i> also occurs and the Mb “Compound I” is recycled to the resting form by using TOP thiol groups as a reducing agent (steps <i>c</i> and <i>d</i>).</p

Maldi-ToF mass spectra of Mb incubated at different conditions: absence of H₂O₂.

<p>(A), the products of a 10 min reaction with H₂O₂, in the presence of 10 mM DBNBS obtained in the absence (B) and in the presence (C) of TOP. Reactions were carried out in 20 mM tris buffer pH 7.4.</p

Catalytic cycle of Mb.

<p>A) Absence of TOP. Electronic absorption spectra of 0.5 µM Mb before (thin solid line),10 min (gray line) and 120 min (thick solid line) after the addition of 5 µM H₂O₂. B) Presence of TOP. Electronic absorption spectra of 0.5 µM Mb before (thin solid line),10 min (gray line) and 35 min (thick solid line) after the addition of 5 µM H₂O₂. C) Effect of TOP concentration on the rate of Mb Compound II recycling to the resting form. After normalization of the spectra of Mb Compound II and recycled Mb by the maximal intensity of Soret band, the rate of oxoferryl Mb recycling to the resting state was calculated by the difference of intensity at 408 nm and the normalized spectra of the recycled Mb. The delta normalized absorbance at 408 nm was divided by the time of recycling and plotted as a function of TOP concentration. Different TOP concentrations were added 10 min after hydrogen peroxide addition (maximal Soret band red shift). Immediately after the formation of Compound II, 5 µM of TOP was added to the medium. These results are representative of a set of two independent experiments with standard deviation of 15%. The reaction was carried out at 37 °C, in 20 mM Tris buffer pH 7.4 treated with Chelex-100®. </p

Experimental EPR spectra of tryptophanyl DBNBS adducts obtained from the reaction mixture containing Mb and H₂O₂.

<p>A) Absence of TOP. Line a and b correspond, respectively, to the EPR spectra of 20 mM DBNBS adduct obtained immediately and 10 min after the addition of 50 µM H₂O₂ to 5 µM Fe³⁺Mb solution. The light gray line, overlapped on line a, corresponds to the spectrum simulated by NLS software, B) Presence of 20 µM TOP: Lines a, b, c and d correspond to the experimental EPR spectrum, simulated composite EPR spectrum, and the simulated rigid and free rotating components, respectively. The spectrum was obtained immediately after the addition of 50 µM H₂O₂ in 5 µM Fe³⁺Mb solution. When present, TOP was previously treated with 1 mM TCEP. EPR conditions were: microwave frequency = 9.5077 GHz, central field, 340 mT, scanning field, 16 mT, number of points, 1024, modulation amplitude, 0.05 mT, gain, 5.0x10⁵, temperature, 293 K, time constant, 0.128 s, scan time, 300 s, microwave power, 20 mW. The reaction was performed in buffer Tris 20 mM previously treated with Chelex-100 ®, pH 7.4, at 25° C.</p

SH content and aggregates of TOP treated with 10 mM (100% reduced cysteines) and 1 mM (40% reduced cysteines) TCEP.

<p>A) WT) wild type TOP, A) + 10 mM TCEP, B) + 10 mM TCEP and 5 µM H₂O₂, C) + 10 mM TCEP and 0.5 µM Mb, D) + 10 mM TCEP and 0.5 µM Mb and 5 µM H₂O₂, E) + 10 mM TCEP and 0.5 µM HRP, F) + 10 mM TCEP and 0.5 µM HRP and 5 µM H₂O₂, A') + 1 mM TCEP, B') + 1 mM TCEP and 5 µM H₂O₂, C') + 1 mM TCEP and 0.5 µM Mb, D') + 1 mM TCEP and 0.5 µM Mb and 5 µM H₂O₂, E') + 1 mM TCEP and 0.5 µM HRP, F') + 1 mM TCEP and 0.5 µM HRP and 5 µM H₂O₂. B) SDS-PAGE of TOP at the following conditions: lanes 1 to 7 correspond, respectively, to molecular weight standard, 100% reduced TOP, + H₂O₂, + Mb, + H₂O₂, and Mb, + HRP, + HRP and H₂O₂. C) SDS-PAGE of 40%-reduced TOP at the following conditions: lanes 1 to 7 correspond, respectively, to molecular weight standard, 40% reduced TOP, + H₂O₂, + Mb, + H₂O₂, and Mb, + HRP, + HRP and H₂O₂. D) This panel shows the large amount of aggregates in aged TOP. The reaction mixtures were incubated for 2 hours in buffer Tris 20 mM Chelex-100 ®, pH 7.4, at 37° and contained 5 µM TOP. The SDS-PAGE of 100% reduced TOP was done with 10 μM TOP.</p

Promising Dual Anticancer and Antimetastatic Action by a Cu(II) Complex Derived from Acylhydrazone on Human Osteosarcoma Models

Osteosarcoma cancers are becoming more common in children and young adults, and existing treatments have low efficacy and a very high mortality rate, making it pressing to search for new chemotherapies with high efficacy and high selectivity index. Copper complexes have shown promise in the treatment of osteosarcoma. Here, we report the synthesis, characterization, and anticancer activity of [Cu(N–N–Fur)(NO3)(H2O)] complex where N–N–Fur is (E)-N′-(2-hydroxy-3-methoxybenzylidene)furan-2-carbohydrazide. The [Cu(N–N–Fur)(NO3)(H2O)] complex was characterized via X-ray diffraction and electron spin resonance (ESR), displaying a copper center in a nearly squared pyramid environment with the nitrate ligand acting as a fifth ligand in the coordination sphere. We observed that [Cu(N–N–Fur)(NO3)(H2O)] binds to DNA in an intercalative manner. Anticancer activity on the MG-63 cell line was evaluated in osteosarcoma monolayer (IC50 2D: 1.1 ± 0.1 μM) and spheroids (IC50 3D: 16.3 ± 3.1 μM). Selectivity assays using nontumoral fibroblast (L929 cell line) showed that [Cu(N–N–Fur)(NO3)(H2O)] has selectivity index value of 2.3 compared to cis-diamminedichloroplatinum(II) (CDDP) (SI = 0.3). Additionally, flow cytometry studies demonstrated that [Cu(N–N–Fur)(NO3)(H2O)] inhibits cell proliferation and conveys cells to apoptosis. Cell viability studies of MG-63 spheroids (IC50 = 16.3 ± 3.1 μM) showed that its IC50 value is 4 times lower than for CDDP (IC50 = 65 ± 6 μM). Besides, we found that cell death events mainly occurred in the center region of the spheroids, indicating efficient transport to the microtumor. Lastly, the complex showed dose-dependent reductions in spheroid cell migration from 7.5 to 20 μM, indicating both anticancer and antimetastatic effects

Oxidative modifications induced by a TR cation radical on cytochrome

<p><b>c</b>.A - Spectral changes of cytochrome <i>c</i> before (light gray line) and after 30 (gray line), and 120 (black line) min UV light irradiation at 254 nm, at pH 4.0. <b>B</b> –The same conditions described for A in the presence of 25 µmol/L TR. <b>C</b> - The same conditions described for Ain the presence of 2.5 mmol/L TR. In this panel, the dashed line represents the original spectrum of cytochrome c before the subtraction of the turbidity sample contribution that resulted in the spectrum shown as a black line <b>D</b> – The effect of different TR concentrations on the rate of Soret band blue shift promoted by UV irradiation. In panel D, the number of asterisks indicates the corresponding spectra shown in panel A (*), B (**), and C (***), from which the data were extracted. The inset shows SEM images and the distribution of particle size obtained for the sample in the condition marked with three asterisks. <b>E</b> – The effect of pH on the rate of Soret band blue shift promoted by UV irradiation in the presence of 25 µmol/L TR. Filled black circles represent the results in the absence of TR and opened circles represent the presence of TR. In panel E, the asterisk in one data point indicates that the point was extracted from the corresponding spectrum shown in panel F. <b>F</b> – The same conditions described for B, at pH 8.0.In this panel, the dashed line represents the original spectra of cytochrome c before the subtraction of the turbidity sample contribution that resulted in the spectrum shown as a black line. Samples containing 3 µmol/L cytochrome <i>c</i> and TR, when indicated, were irradiated in a 5 mmol/L phosphate buffer at 25°C in the cuvette with a 4 mW/cm^-2 as function of 254 nm of excitation at a distance of 4 cm from 1cm² of a selected sample area.</p