The effects of LS081 on ferritin expression were determined under two conditions: RPMI1640-10% FCS to which 2 μM ferric ammonium citrate was added or RPMI with 10% iron saturated FCS. As shown in Figure 2, LS081 at 3 and 10 μM stimulated ferritin synthesis from both ferric ammonium citrate and iron saturated Tf. In preliminary
experiments the level of ferritin protein was not significantly increased by compound alone (data not shown). Figure 2 The effect of LS081 on ferritin expression. PC-3 cells were treated for 16 hr with DMSO alone, or 3 or 10 μM LS081 in the presence of non-transferrin-bound-iron selleck products (ferric ammonium citrate, left panel) or transferrin-bound-iron (Fe-saturated-Tf, right panel). The cellular proteins were separated by SDS-PAGE, and ferritin heavy chain, and β-actin detected by Western blotting as described Pitavastatin in the Methods. The top panel shows a representative autoradiography. The bottom panel shows the ratio of ferritin to the actin loading control by densitometric analysis (mean values ± SEM of 3-4 separate experiments). *: p < 0.05, **: p < 0.01 compared
to DMSO alone by 1-way ANOVA with Tukey’s posttests. Iron LCZ696 facilitation is cytotoxic to cancer cells We examined the effect of the iron facilitator LS081 on ROS generation using DCFDA whose fluorescence intensity is increased in response to elevated intracellular ROS. As shown in Figure 3, K562 cells had significantly increased levels of ROS production when exposed to LS081 in the presence of ferric ammonium citrate but not with iron or LS081 alone. Figure 3 The effect of LS081 on ROS generation. Approximately 5 × 105 K562 cells were treated for 30 min with 0.1% DMSO alone, 10 μM ferric ammonium citrate alone, 3 or 10 μM LS081 alone, or the combination of Fe and LS081 at the indicated concentrations. The cells were then incubated with DCFDA and fluorescence measured by a BD Calibur Flow cytometer expressing the fluorescence as the mean total fluorescence intensity in the gated area. Shown are the means ± SEM of 3 separate
experiments with 2-3 replicates for each experiment. *** denotes P < 0.001 compared to the DMSO, Fe, or LS081 alone by 1-way ANOVA with Tukey's Non-specific serine/threonine protein kinase posttests. The proliferation of PC-3 cells, a prostate cancer cell line, was not inhibited by 10 μM ferric ammonium citrate or 10 μM LS081 when cultured in 10% FCS-RPMI1640 for 24 or 48 hrs (Table 1) or 72 hr (data not shown). However, as also shown in Table 1, treatment with 10 μM LS081 plus 10 μM ferric ammonium citrate for 24 hr or 48 hr significantly reduced the number of cells relative to controls. When grown in serum-free medium (Figure 4), 267B1 cells, an immortalized, non-malignant prostate cell line, showed slight growth inhibition with 3 or 10 μM LS081 alone with no potentiation of growth inhibition by the addition of 2 μM ferric ammonium citrate.