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  • Vaborbactam br Fig Mitochondrial superoxide O generation det

    2019-10-21


    Fig. 6. Mitochondrial superoxide (O2%−) generation determined after 6 h treatment with investigated compounds applied at 50 μM (MCF-7 cells) and 75 μM (AsPC-1 cells). Analyses have been Vaborbactam performed by means of MitoSox Red staining. Results are represented as percentages of Vaborbactam positive for O2%− production and over median fluorescent intensity (MFI) expressed in arbitrary units (AU) computed for O2%−-positive subpopulation. All results are represented as the mean ± SD of two replicates from 3 independent experiments.
    (0.81 ± 0.05-fold). Nevertheless, results on AsPC-1 spheroids disclose an interesting similarity in pattern of 1 and CDDP activities (Figs. 9 and 11). As reviewed above, CDDP does not induce cell death in AsPC-1 2D cells over 24 h of incubation [116]. On the contrary, CDDP against AsPC-1 3D model displays an activity at all 3 tested concentrations (Figs. 9 and 11). In samples subjected to CDDP at 100 μM, extreme regression in size is recorded (0.4 ± 0.1-fold on the day 8). CDDP at 10 μM suppresses growth of spheroids (1.0 ± 0.1-fold on the day 8), while tumors treated with 1 μM record a growth of 1.4 ± 0.1-fold. Opposite to results on MCF-7 3D model, 1 was more effective on AsPC-1 tumors than CDDP (Figs. 9–11). Complex 1 at 100 μM interrupted spheroidal assemblage quite promptly after its addition on AsPC-1 3D tumors (Fig. 11). Initially, such a process produced erroneous im-pression of gain in size, while after 6 days of treatment spheroids are completely destroyed. In those samples, residual clustering of cells could be observed, but those formations were less compact compared to 
    the one seen after CDDP treatment at the same concentration. The reason why the disintegration of the spheroidal structure appears so quickly after 1 was added, as AsPC-1 tumors, may be due to its pro-apoptotic activity, but the possibility that 1 induces CSCs phenotype reprogramming with consequential loss of cells' ability to congregate remains to be further investigated.
    3.5. DNA binding studies
    As reviewed above, results of the current biological assessment in both AsPC-1 and MCF-7 cells treated with 1 revealed that the me-chanism of this compound's activity is most probably related to de-rangement of DNA replication process. However, the differences in cell cycle changes at the first place, together with variable pattern in cas-pases activation and MTP dissipation between 1 and CDDP, strongly implied that those two compounds do not interrupt cellular homeostasis
    Fig. 7. Dissipation of mitochondrial transmembrane potential (MTP) in non-treated and treated AsPC-1 cells after 6 h incubation with investigated compounds applied at 75 μM. In the upper panels cells are discriminated according to the type of cell death (non-stained viable cells and stained with Annexin V and/or 7‑AAD). In the middle panels cells are discriminated according to staining with MitoSense Red dye (negative cells in lower left and right quadrants have scattered MTP) and concomitant staining with Annexin V (apoptotic cells in upper right and lower right quadrants). In the lower panels cells are discriminated according to staining with MitoSense Red dye and concomitant staining with 7‑AAD (cells in necrosis or advanced phase of apoptosis in the upper right or lower right quadrants). All results are represented as the mean ± SD percentages of two replicates from three independent experiments.
    Fig. 8. Dissipation of mitochondrial transmembrane potential (MTP) in non-treated and treated MCF-7 cells after 6 h incubation with investigated compounds applied at 50 μM. In the upper panels cells are discriminated according to the type of cell death (non-stained viable cells and stained with Annexin V and/or 7‑AAD). In the middle panels cells are discriminated according to staining with MitoSense Red dye (negative cells in lower left and right quadrants have scattered MTP) and concomitant staining with Annexin V (apoptotic cells in upper right and lower right quadrants). In the lower panels cells are discriminated according to staining with MitoSense Red dye and concomitant staining with 7‑AAD (cells in necrosis or advanced phase of apoptosis in the upper right or lower right quadrants). All results are represented as the mean ± SD percentages of two replicates from three independent experiments.