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  • br DMSO D O and the

    2020-08-18

    
    DMSO:D2O (3:2) and the stability in these solutions was higher (4 days). Both Titanocref (2) and Titanofin (4) are soluble in DMSO:PBS (1:99) in the micromolar range (necessary for the biological assays).
    2.2. Cytotoxicity, selectivity, cell death and Erythromycin lactobionate arrest
    The cytotoxicity of the bimetallic [(h-C5H5)2TiMe(m-mba) Au(PR3)] (PR3 ¼ PPh3 2 Titanocref and PEt3 4 Titanofin), the two monometallic gold derivatives [Au(Hmba)(PR3)] (PR3 ¼ PPh3 1 cref; PEt3 3 fin), and monometallic titanocene dichloride was evaluated. For comparative purposes, the cytotoxic profile of Auranofin was also determined. In this assay, human clear-cell renal carcinoma Caki-1 cells and non-tumorigenic human fetal lung fibroblasts (IRM-90) were incubated with the compounds 1e4, Auranofin or titanocene dichloride for 72 h. The results are summarized in Table 2. In the supplementary information section there is a more complete table that includes IC50, IC20, and IC10 values at 72 h and IC50 and IC20 values at 24 h (Table S3).
    The heterometallic compounds are considerably more toxic to the renal cancer cell line (Caki-1 cells) than titanocene dichloride and Auranofin (in the sub-micromolar range as already reported for Titanocref for different renal cancer cell lines [26,27]).
    In addition, Titanocref 2 is far more toxic in the nanomolar range than the monometallic gold compound 1 cref Erythromycin lactobionate on these cells. Tita-nofin 4 is three times more toxic than the monometallic compound with PEt3 (3) fin. This supports the hypothesis that the presence of two biologically active metals improves cytotoxicity. Importantly, both heterometallic compounds are more selective to renal cancer cells than Auranofin or the monometallic gold compounds, espe-cially Titanocref 2.
    In addition to the IC50 at 72 h, we also assessed the IC50 after 24 h of incubation (Table S3 in SI). Because we sought to determine the changes in behavior as well as their associated molecular signaling it was essential that the cells be alive, therefore we used concen-trations at which we know that after 72 h incubation 80% (IC20) or 90% (IC10) of the cells would be viable (see Table S3 in SI). Some assays were carried out after 24 h of incubation with the 72 h IC20 which is the lowest dose at which we saw after 24 h detectable changes in expression levels of the proteins we interrogated (Table S3 in SI). The relevance of the 24 h IC50 evaluation is to demonstrate that the 72 h IC20 of the compound do not induce significant cytotoxicity after 24 h incubation.
    Following the evaluation of the cytotoxicity of bimetallic Tita-nocref (2), Titanofin (4) and monometallic gold Auranofin, we proceeded to evaluate how the cells died. For this assay Caki-1 cells were incubated with the indicated compound at the IC50 concen-tration for 72 h. We observed that all the compounds induce
    Table 2 Cell viability IC50 values (mM) for Caki-1 cells and IMR-90 fibroblasts treated with bimetallic Titanocref (2), Titanofin (4) and monometallic gold cref (1) and fin (3) compounds. Monometallics Auranofin (AF) and titanocene dichloride (TDC) were used as controls.a The IC50 are reported with the standard deviation of the sample mean.
    Compound
    a All compounds were dissolved in 1% of DMSO and diluted with media before addition to cell culture medium for a 72 h incubation period. Cisplatin was dissolved in H2O. Structures of the compounds are provided in Chart 1. b Values reported in reference 30.
    Fig. 2. Effects of Titanocref (2), Titanofin (4) and Auranofin on apoptotic index and cell cycle distribution on Caki-1 cells. (A) Cell apoptosis in bimetallic Titanocref (2), Titanofin (4) and monometallic Auranofin treated cells was detected by Annexin V and propidium iodide (PI) labeling detected by flow cytometry after 72 h of incubation with IC50. The extent of apoptosis was determined by the percentage of the total cells that were positive for PI reported in each quadrant 2 (Q1 ¼ dead cells, Q2 ¼ Apoptotic cells, Q3 ¼ Alive cells). Treatment with of bimetallic Titanocref (2), Titanofin (4) and monometallic Auranofin for 24 h caused cell cycle arrest. Cell-cycle analysis was conducted by FxCycle staining and flow cytometry 24 h after treatment with bimetallic Titanocref (2), Titanofin (4), monometallic Auranofin or DMSO (0.1%) control. Numbers in the bar-graph (C) correspond to histograms (D) indicate the percentage of cells in the Sub G1, G0-G1, S and G2-M phases of the cell cycle (values below 3% were not reported on the bar-graph).
    apoptosis to some degree at their IC50 concentration. For the TieAu compounds Titanocref (2) and Titanofin (4) (at nanomolar con-centration) 58% and 76% of cells die by apoptosis (Fig. 2).
    Auranofin and other gold (I) compounds are known to induce apoptosis in several cancer cell lines [35,42], TDC also induces apoptosis albeit at much higher concentrations than the afore-mentioned compounds [43]. Thus the apoptotic phenotype of bimetallic Titanofin and Titanocref may be due to the presence of both, the Ti and Au fragments.