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  • br NMR spectroscopic investigations at ambient temperature r

    2019-10-21


    NMR spectroscopic investigations at ambient temperature revealed evidence consistent with the presence of just one Cd(II) complex species in solution of 1. However, existence of one set of signals in NMR spectra may also correspond to the pentacoordinated mononuclear complex obtained by the cleavage of the two chloride bridges that give rise to the dinuclear complex 1. In order to address this issue experimentally ob-tained UV–Vis spectrum of 1 in DMSO (Fig. S12, Supplementary ma-terial) was compared to the calculated MK-571 sodium salt hydrate spectra of free aphaOMe ligand, pentacoordinated mononuclear complex and di-nuclear complex 1 (Fig. S13, Supplementary material). Theoretical spectrum of dinuclear species has strong absorption at 310 nm (H-1 → LUMO) with shoulder band at 265 nm (H-1 → L + 2), and shows the best agreement with the experimental data. As a consequence of change in the coordination environment, mononuclear Cd complex has addi-tional transition at 211 nm (Fig. S13, Supplementary material). All absorptions can be assigned to the intraligand π–π* transitions as it is indicated by composition of the main molecular orbitals relevant for the electronic transitions (Fig. S14, Supplementary material).
    3.4. Anticancer investigations
    3.4.1. Superior pro-apoptotic activity of 1 as compared to CDDP Pro-apoptotic activity of 1 has been assessed after 24 h incubation
    on MCF-7 and AsPC-1 cells by means of Annexin V/PI dual staining method. Percentages of Annexin V single-stained and double-stained cells were summarized for each concentration of investigated com-pound. The computed percentages were charted against corresponding concentrations and ApoC50 concentration was computed as the one that corresponds to 50% of whole apoptotic events (early and late apoptosis) on the concentration-response curve (Fig. S15, Supplementary mate-rial). Our experimental result reveals a high incidence in apoptotic death induction and this for both cell lines (Fig. 3, Table 3). Activity of 1 is especially powerful on MCF-7 cells as at 30 μM all cells are Annexin V/PI positive (Fig. 3). AsPC-1 cells, notorious for broad-spectrum drug resistance [82,83], are responding surprisingly well to the treatment with 1 as, at 30 μM more than half of AsPC-1 cells are either in early or advanced apoptosis. Comparing current with our previously published data [99,116–118] here investigated complex 1 is the strongest inducer of apoptosis in AsPC-1 cells, including CDDP. The only metal complex that induced apoptosis in this CSC model reached significant percentage of Annexin V-stained cells only at 75 μM [99], as well as the best one referring to organic compounds [116].
    As mentioned before, activity of CDDP on AsPC-1 cells has been evaluated in our previous work with a very poor outcome [116]. In the current study, CDDP reveals to be more effective on MCF-7 than AsPC-1 cells, but its pro-apoptotic activity was still less remarkable compared to 1 (Fig. 3). While more than a half of MCF-7 cells in the samples subjected to 1 at 10 μM were found as double-stained, CDDP at 75 and  Journal of Inorganic Biochemistry 190 (2019) 45–66
    100 μM guided only one third of those cells toward advanced stages of apoptotic death. Relying on results presented here and those we pre-viously reported [116–119], 1 showed superior activity on both cell lines in respect to CDDP or other compounds investigated on CSCs.
    3.4.2. Changes in cell cycle distribution indicate on DNA replication issue The difference in intensity of apoptotic response between MCF-7 and AsPC-1 cells could be originated by either 1 striking different cel-lular targets in each cell line or by the fact that those cell lines from different phenotype respond in a differential mode to the same chal-lenge. Although changes in distribution within mitotic phases cannot provide more precise information on the mechanism of drug's activity, those can at least indicate which process within the cell cycle was mostly distressed by 1. In Fig. 4, noticeable concentration-dependent shifts in distribution of cells are observed within mitosis when MCF-7 cells are subjected to 1. A slight accumulation in the S phase remains the dominant alteration in cells treated with 10 μM, progressing to a mitotic arrest in S phase at 30 μM. Within concentration from 50 to 100 μM, 1 induces G0/G1 blockage with a magnitude increasing in concentration-dependent manner. Such a rearrangement denotes on