Paraptotic Cell Death as an Unprecedented Mode of Action Observed for New Bipyridine-Silver(I) Compounds Bearing Phosphane Coligands

In this work, we investigated the anticancer activity of several novel silver(I) 2,2′-bipyridine complexes containing either triphenylphosphane (PPh3) or 1,2-bis(diphenylphosphino)ethane (dppe) ligands. All compounds were characterized by diverse analytical methods including ESI-MS spectrometry; NMR, UV–vis, and FTIR spectroscopies; and elemental analysis. Moreover, several compounds were also studied by X-ray single-crystal diffraction. Subsequently, the compounds were investigated for their anticancer activity against drug-resistant and -sensitive cancer cells. Noteworthily, neither carboplatin and oxaliplatin resistance nor p53 deletion impacted on their anticancer efficacy. MES-OV cells displayed exceptional hypersensitivity to the dppe-containing drugs. This effect was not based on thioredoxin reductase inhibition, enhanced drug uptake, or apoptosis induction. In contrast, dppe silver drugs induced paraptosis, a novel recently described form of programmed cell death. Together with the good tumor specificity of this compound’s class, this work suggests that dppe-containing silver complexes could be interesting drug candidates for the treatment of resistant ovarian cancer.


S3. Single crystal X-ray crystallography [Ag(2,2'-bipy)(PPh3)][CF3SO3] (1)
A total of 637 frames were collected.The integration of the data using a triclinic unit cell Refinement using the space group P-1, with Z = 2 for the formula unit, C29H23AgF3N2O3PS.The final anisotropic full-matrix least-squares refinement on F 2 with 361 variables converged at R1 = 2.68%, for the observed data and wR2 = 5.65% for all data.The goodness-of-fit was 1.119.The largest peak in the final difference electron density synthesis was 0.608 e -/Å 3 and the largest hole was -0.443 e -/Å 3 with an RMS deviation of 0.081 e -/Å 3 .On the basis of the final model, the calculated density was 1.655 g/cm 3 and F(000), 680 e -.

[Ag(4,4'-CH3-2,2'-bipy)(PPh3)][CF3SO3] (2)
A total of 2418 frames were collected.The integration of the data using a monoclinic unit cell yielded a total of 165841 reflections to a maximum θ angle of 30.53° (0. for all data.The goodness-of-fit was 1.132.The largest peak in the final difference electron density synthesis was 2.579 e -/Å 3 and the largest hole was -1.890 e -/Å 3 with an RMS deviation of 0.091 e -/Å 3 .On the basis of the final model, the calculated density was 1.602 g/cm 3 and F(000), 744 e -.

Figure
Figure S34. A. LC-DAD chromatogram obtained for compound 1 in MeCN: showing the two complexes identified in Figure S30.

Figure S36 .
Figure S36.Impact of oxaliplatin resistance on 1, 4 and 7 in comparison to oxaliplatin.HCT116 subclones were treated with increasing concentrations of the test compounds and the cytotoxicity was evaluated by MTT assays after 72 h.The graph shows dose-response curves of one representative experiment performed in triplicates.

Figure S37 .Figure S38 .Figure S39 .
Figure S37.Anticancer activity of novel silver compounds in colonic epithelial fibroblasts.F331 cells were treated with increasing concentrations of the compounds for 72 h, followed by MTT assay.The data represented in the graphs are the mean ± SD of three independent experiments, each performed in technical triplicates.

Figure S40 .
Figure S40.Impact of the tested compounds on cell cycle distribution of SK-OV-3 (A) and MES-OV (B) cells after 24 h of treatment.The percentage of cells in G0-G1, S and G2/M phase were analyzed in ethanol-fixed cells with propidium iodide (PI) followed by flow cytometry after 24 h treatment.The mean ± SD was derived from three independent experiments.

S35Figure S41 .
Figure S41."DNA ladder" pattern of DNA fragments occurring during apoptosis was evaluated after 24h treatment with the indicated concentrations of silver compounds.As positive control 250 and 500 µM H 2 O 2 was used to treat the cells for 24 h.

Figure S42 .
Figure S42.Cytoplasmatic vacuoles indicative for paraptotic cell death induced by silver compounds treatment.Phase-contrast images of MES-OV cells treated with the indicated compounds for 6 h (A) and 24 h (B) (200× magnification, scale bar: 100 μm).

Figure S43 .Figure S44 .
Figure S43.Spinning disk confocal microscopy of MES-OV cells treated with the indicated compounds and concentrations for 24 h.Representative pictures were taken in confocal mode, Z-stack and max intensity projection (192x magnification and 60x objectives 600-x magnification) of vesicles with stain of the ER (ERTracker in red), DIC (Differential interference contrast) and mitochondria (MitoTracker in green).

Figure S45 .Figure S46 .
Figure S45.Impact of caspase inhibition on vacuole formation.Phase-contrast images of MES-OV cells treated with the indicated drugs in combination with and without the pan-caspase inhibitor Z-VAD-FMK for