Rhodacyanine Derivative Kills Resistant Cancers Figure 1. MKT-077 derivative, YM-1, shows enhanced toxicity and altered localization. MCF7 cells were treated for 24 hours with three concentrations of MKT-077 or YM-1. After 24 hours, medium was collected and analyzed by LDH assay. Values shown are a % of vehicle treatment 6 SD (A). MCF7 cells were treated with MKT-077, YM-1 or methylene blue (MB). Mitochondrial fractions were collected and compound location was measured by spectrophotometer (B). doi:10.1371/journal.pone.0035566.g001 released by the associated toxicity. The other cancer cell lines tested all displayed toxicity following YM-1 administration; whereas, the two immortalized cell lines displayed minimal to no toxicity by LDH assay (Figure 2B). This demonstrated that the cytosolic localization of YM-1 did not affect its specificity for cancer cells. YM-1 efficacy was then tested in a cell model of tamoxifen- resistance. The toxicity of YM-1 in a refractory tamoxifen (4- OHT) resistant MCF7 (TR-MCF7) cell line was compared to that of the parental MCF7 (non-resistant) cell line. Indeed, YM-1 effectively killed both standard and resistant (TR-MCF7) cells after 48-hour incubation (Figure 3A). Given the previous concerns with chronic MKT-077 treatment, we speculated that a shorter treatment with YM-1 might be equally toxic. To test this, MCF7 cells and TR-MCF7 cells were treated with 10 mM YM-1 for 4 hours. This was removed and replaced with vehicle for 44 hours. In addition, TR-MCF7 cells were treated with either 4-OHT or the vehicle for 4-OHT (95% EtOH) (Figure 3B). In each case, minimal toxicity was observed. Cell viability (MTT) assays were then used to test whether this shorter treatment strategy was affecting cell proliferation. The TR- MCF7 cells were grown in media containing 10 mM 4-OHT. Our designed treatment strategy contained four conditions all termi- nating at 48 hours: 1. 10 mM 4-OHT alone for 48 hours, 2. YM-1 (or vehicle) treatment for 4 hours followed by re-addition of 10 mM 4-OHT for 44 hours, 3. YM-1 (or vehicle) treatment for 4 hours followed by 95% EtOH (vehicle for 4-OHT), and 4. YM-1 (or vehicle) treatment for the full 48-hours. MTT assays revealed that the 4-hour 10 mM YM-1 followed by 10 mM 4-OHT treatment reduced viability by 60% relative to the 4-OHT treatment alone. The 10 mM YM-1 followed by 95% EtOH treatment did not alter viability (Figure 4A). The 48-hour 10 mM YM-1 treatment reduced viability by 40% compared to 48-hour 4- OHT treatment, similar to Figure 3A. All treatment strategies containing YM-1 were analyzed by two- way ANOVA (Figure 4B). This analysis revealed a significant effect by treatment strategy and concentration of YM1 (F(4, 30) = 41.04, p,0.0001), (F(2,30) = 54.22, p,0.0001). The interac- tion between treatment strategy and concentration was not significant (F(8,30) = 1.83, p = 0.1107). Bonferroni post-hoc anal- ysis of this 2-way ANOVA showed no significant differences between any of the concentrations used for the 48-hour YM-1 PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35566