treatment conditions, with the inclusion of the 4-OHT 48 hour treatment and vehicle treatments as separate groups, were analyzed by one-way ANOVA (F,(5,12) = 24.33, p,0.0001). Tukey’s post-hoc test revealed that 4 hour YM-1 followed by 4- OHT was significantly different from the 4-OHT 48 hours treatment (p,0.05), whereas both the 48 hour 10 mM YM-1 and the 4 hour 10 mM YM-1 followed by 95% EtOH treatments were not significantly significant from the 4-OHT 48 hour treatment (Figure 4F). This analysis also displayed that 10 mM YM1 followed by 4-OHT is significantly different from 10 mM YM1 followed by 95% EtOH (p,0.05). These findings suggested that just a 4 hour treatment of 10 mM YM-1 could re-sensitize TR-MCF7 cells to tamoxifen/4-OHT, stopping cell growth without causing overt toxicity. The potential mechanisms for this phenomenon were then explored. One plausible mechanism was aberrant kinase activity, which is known to promote tamoxifen resistance by phosphorylating ERa at a site known to promote estrogen independent activity [14,15,16,17,18]. We treated TR-MCF7 cells as described for the experiments in Figure 4A & B. Nuclear proteins were isolated and probed for levels of ERa pS167, a site that when phosphorylated conveys tamoxifen independence. Indeed, phosphorylation of ERa pS167 was elevated in the presence of 4-OHT; however, the addition of 10 mM YM-1 abrogated this event (Figure 5A & B). YM-1 did not alter the nuclear localization of ERa into the nucleus (Figure 5C & D). Rhodacyanine Derivative Kills Resistant Cancers S167 of ERa falls is contained within an Akt (Akt/PKB) consensus site. Akt is a pro-survival kinase with two isoforms known to interact with ERa. We treated MCF7 cells with 10 mM YM-1 for 6 hours to avoid toxicity and looked for changes in either Akt levels or activation. The levels of Akt1 and Akt2 were dose dependently reduced by YM-1 (Figure 6A & B). This suggests that YM-1 can cause toxicity specific to cancer cells, similar to the parent compound MKT-077, but that YM-1 does so by reducing pro-survival kinases like Akt, potentially leading to alterations in resistance mechanisms in refractory tumors. This data agrees with work previous work demonstrating that the effects of LY294002, an inhibitor of the PI3K/Akt signaling pathway inhibitor, on tamoxifen-induced apoptosis were specific for inhibiting Akt activity [16]. Discussion Here we describe the therapeutic potential of an MKT-077 derivative, YM-1. This compound, similar to MKT-077, was specifically toxic to cancer cells. YM-1 also had greater efficacy and cytosolic localization than MKT-077. Brief exposure to YM-1 was able to re-sensitize refractory breast cancer cells to tamoxifen, a common therapy used in the clinic. This mechanism was shown to be Akt dependent as YM-1 was able to reduce Akt levels as well as the phosphorylation of ERa at an Akt consensus site. These data demonstrate the potential for rhodacyanine derivatives in the treatment of refractory cancers. Figure 5. Phosphorylation but not localization of Estrogen Receptor a altered by YM-1. TR-MCF7 cells were treated with indicated conditions for 4 hours. Nuclear isolates and cytosolic fractions were compared by Western blot, representative blots shown (A & C). Densitometry analysis of pERa and ERa levels displayed as % of vehicle treatment 6 SD (B & D). doi:10.1371/journal.pone.0035566.g005 PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35566