Figure 6

The activity of HQBA is reversed by iron. (a) AGS, PA-1 and DLD-1 cells were incubated in the presence of 9.8 nM to 20 μM HQBA±0.5 M equivalents of ferrous ethylenediammonium sulfate tetrahydrate for 72 h in 96-well plates. Cells were then lysed and viability was assessed by measuring endogenous lactate dehydrogenase activity. (b) STF3A cells were plated in 96 well format and wells were treated with 9.8 nM to 20 μM HQBA±0.5 M equivalents of ferrous ethylenediammonium sulfate tetrahydrate for 18 h. Luciferase activity in lysates was measured and normalized to endogenous lactate dehydrogenase activity. Dose curves were plotted with Graphpad Prism (La Jolla, CA, USA). (c) DLD-1 cells were plated in 24-well plates in the presence of the indicated concentration of FeCl₃, and allowed to grow for 24 h. Cells were washed three times with phosphate-buffered saline and then incubated with 500 nM HQBA for an additional 72 h. Viability was measured as described. (d) DLD-1 cells were grown in the presence of 20 μM FeCl3 for 24 h, washed twice with phosphate-buffered saline, and then treated with the indicated concentration of HQBA in dimethyl sulfoxide for an additional 24 h before lysis and measurement of AXIN2 mRNA levels by quantitative reverse transcriptase–PCR. Data are shown as ±s.d. and significance calculated by paired t-test.