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  • br Fig Changes in size and

    2019-10-21


    Fig. 10. Changes in size and morphology of MCF-7 3D spheroids over the 8 days of treatment with investigated compounds. Images have been taken every other day on Celigo imaging cytometer using Celigo software. Scale bar: 200 μm.
    EB–CT-DNA system upon addition of 1 does not decrease > 50%, which is generally accepted threshold value if compound interacts with DNA by intercalation [155], it can be concluded that 1 binds to DNA in a different mode from EB. Fig. 12B shows the characteristic emission spectrum of H when it is bound to CT-DNA. The addition of 1 caused appreciable reduction in the fluorescence intensity of H–CT-DNA system in a concentration de-pendent manner. The quenching of H–CT-DNA showed an initial sa-turation at 10 μM concentration of 1 (inset in Fig. 12B). It is important to note that the fluorescence intensity was reduced to nearly half of the initial value at this concentration of 1. The half-reciprocal plot of the quenching data according to Stern-Volmer (see Supplementary data) resulted in a linear plot (Fig. 12C) with a quenching constant Ksv = 3.27. The value is comparable with competition similar values obtained with other metal complexes binding to minor groove [156]. With increasing concentration of 1, an additional decrease in fluores-cence intensity was observed, followed by saturation in competitive displacement from H–CT-DNA system. The decrease of H-CT-DNA fluorescence intensity was 63% with maximal applied concentration of 1. The obtained results of the extent of the fluorescence quenching of EB by competitive displacement from EB–CT-DNA system and groove binder H from H–CT-DNA system demonstrated that 1 is a minor groove binder.
    The DNA interaction study show non-covalent low binding strength of 1 into DNA minor groove together with the lack of DNA cleavage in 
    pUC19 experiment. The changes in AZD8931 distribution signify that 1 interferes in the process of DNA replication, while comparative analysis indicates that its mechanism of action differs from the one of CDDP (vide supra). Taking into account the high affinity of Cd(II) for S ligands, like thiol groups of enzymes and proteins [113], possible molecular target of 1 is rather related to protein(s) involved in the control of re-plication than DNA itself.
    Molecular docking showed that the compound 1 preferentially binds to the minor groove of DNA (Fig. 13). Out of 20 docking solu-tions, the best 12 positioned compound 1 near or into the minor groove, and only 3 solutions placed compound 1 in the major groove. Compared to the HSA binding (vide infra), the ChemPLP docking score for 1-DNA interaction was significantly smaller (−52.67 compared to −83.50; Table S2, Supplementary material). This is in line with the experimental findings that 1 binds with higher affinity to the proteins (HSA, vide infra) than to DNA.
    3.6. Acute lethality assay
    As a preliminary toxicity screening, in vivo acute lethality of 1 and reference compound CDDP were tested on brine shrimp Artemia salina after 24 h incubation. LC50 value for complex 1 (0.316 ± 0.007 mM) was significantly higher than for CDDP (0.006 ± 0.004 mM), which
    Fig. 11. Changes in size and morphology of AsPC-1 3D spheroids over the 8 days of treatment with investigated compounds. Images have been taken every other day on Celigo imaging cytometer using Celigo software. Scale bar: 200 μm.
    indicates that CDDP induced higher incidence of lethality in compar-ison to 1. Since that there is a good correlation between the results of LC50 obtained in the current bioassay and the results of the Acute Oral Toxicity Assay in Mice [157], it can be anticipated that 1 would possess the lower acute toxicity in comparison to CDDP.
    3.7. Interaction with HSA
    3.7.1. Experimental study of interaction between 1 and HSA
    HSA is well known for its binding capacity and repository for an extraordinarily diverse range of molecules which makes it an important factor in the pharmacokinetic behavior of many drugs by affecting their efficiency and rate of delivery. Because of this, the studies of interac-tions between potential anticancer drugs and HSA as a potential drug carrier are of great importance in cancer science. Fluorescence spec-troscopy proved to be useful for characterization of small molecule - HSA interactions. Aromatic amino acids Tyr, Phe and Trp can emit light by fluorescence upon excitation with 280 nm light, and among them Trp has the largest quantum yield. The HSA has only one Trp residue (Trp214) which is close to two binding sites where the majority of drug molecules bind (Sudlow site I and II). Upon binding of a small molecule