Figure 3.

FDA-approved drug hits promote cisplatin toxicity and stimulate DNA adduct formation in resistant IGROV-CP20 tumor cells. (A) Representative images of live/dead assay (see Section 4) showing the effect of 50 µM cisplatin (Pt) on the cell viability of IGROV or IGROV-CP20 cells, as indicated. Pretreatment with 10 µM Tranilast, Telmisartan, or Amphotericin B for 24 h increased cell death of IGROV-CP20 cells when combined with cisplatin. (B) Quantification of experiments shown in panel A reveals a significant decrease in viability of IGROV-CP20 cells treated with a mixture of cisplatin and indicated drugs (n = 10 fields; *** p < 0.001, ANOVA). (C) Pt adducts were evaluated via dot immuno-blot (see Section 4) with DNA samples from IGROV cells (above the dashed line) and IGROV-CP20 cells (below the dashed line), which were spotted as indicated in the membrane map (on the left). Two dot blot images demonstrate that cisplatin (Pt) in association with Tranilast, Telmisartan, or Amphotericin B induces a significant increase in the DNA adduct signal compared to the IGROV-CP20 treated with cisplatin alone (D) The graph shows quantification of the DNA adduct signal in dot blot experiments (n = 10 fields; ** p < 0.01, * p < 0.05, ANOVA). (E) Cells were treated as in panel A and prepared for ICP-MS (see Section 4) to evaluate intracellular platinum levels. Only the combination of Amphotericin B with cisplatin led to an increase in the overall platinum levels in the cells. Sensitive IGROV cells (green bars) were used as a positive control for cisplatin treatment. The Pt content of each specimen was normalized to the total cell numbers as nM of PT in 10⁶ cells (n = 3 experiments; *** p < 0.001, ANOVA). Scale bar: 320 µM (A).