High through-Put Screening of Chemicals That Stimulate Iron Uptake: A Novel Approach to Discovery of Anti-Cancer Drugs

Iron (Fe) is an essential nutrient required for growth of all cells. Actively growing cells appear to have a greater need for iron at least as measured by increased expression of the transferrin receptor (TfR), the major route by which iron enters cells. Various attempts have been made to use iron chelators to deplete cells of iron as a means for decreasing the growth of cancer cells. We hypothesized that a contrary approach of enhancing iron entry would allow for generation of increased reactive oxygen species which could then potentiate radiation and radiomimetic drugs. To identify small molecules that would accelerate iron uptake we used a high through-put screening system in conjunction with a reporter system of K562 cells loaded with the divalent metal chelator calcein whose fluorescence is quenched with chelation of Fe 2+ . Small molecules that stimulate Fe uptake were defined as causing an increase of calcein fluorescence quenching compared to Fe alone. K562 cells were exposed to 0.1 μM calcein for 10 minutes, thoroughly washed, and 1 × 10 5 cells plated into each well of multiple 96-well plates. After equilibration of the plates at 37° C, aliquots of the individual components of an in-house chemical library of ~10,000 compounds dissolved in DMSO were screened in duplicate or triplicate and fluorescence measurements made at 0 and 30 min after addition of 10 μM FeNH 4 SO 4 in a Synergy IV plate reader. 30 chemicals were identified that stimulated iron-induced quenching of calcein fluorescence. The stimulation was verified by dose response curves and the lack of toxicity noted by having no significant cell death after exposure for up to 5 days. One of the compounds, LS-0108076, at 3 or 10 μM stimulated iron uptake from FeNH 4 SO 4 by 50% in K562 cells. LS-0108076 also facilitated uptake from transferrinbound iron in a prostate cancer line as evidenced by a 2-fold increase in ferritin levels when PC-3 cells were cultured with 10% serum. LS-0108076 (3μM) also significantly increased generation of ROS from 100±0% to 150±18% as measured with DCFDA but only in the serum-free Hepes buffer with 2 μM FeNH 4 citrate. LS-0108076 was further examined in prostate cancer and normal prostate cells for the ability to enhance cell killing by bleomycin, a radiation mimetic anti-cancer drug that chelates Fe and causes cell death by interchelating with DNA and producing DNA strand breaks. In both the PC3 and DU145 prostate cancer cell lines, LS-0108076 enhanced bleomycin cell killing in serum-free medium with further potentiation by the addition of 2 μM Fe 3+ to LS-0108076. Similarly, LS-0108076 potentiated radiation induced cell death with the effects also enhanced by the presence of Fe 3+ and with marked increase of DNA strand breaks as detected by a comet assay. In normal prostate cells LS-0108076 had no affect on bleomycin cell toxicity. In summary, we have developed a high through-put screening technique that identified small molecules that stimulate iron uptake and demonstrated that compounds which facilitate iron uptake increase cell sensitivity to radiation therapy and chemotherapy and can be used to potentiate anti-cancer agents.