Rhodacyanine Derivative Kills Resistant Cancers Figure 3. TR-MCF7 cells and MCF7 cells susceptible to YM-1 toxicity at 48 hours but not at 4 hours; tamoxifen does not alter cytotoxicity. TR-MCF7 and parental MCF7 cells were treated for 48 hours in with 10 mM YM-1. After 48 hours, media were collected and analyzed by LDH assay. Values shown are a % of vehicle treatment 6 SD (A). MCF7 cells were treated for 4 hours with 10 mM YM-1. At 4 hours, medium was replaced with standard growth media for 44 hours. TR- MCF7 cells were treated with 10 mM YM-1 for 4 hours. At 4 hours, the media was removed and replaced with standard TR-MCF7 media containing 10 mM 4-OHT or 95% EtOH, the vehicle for 4-OHT, for 44 hours. After 48 hours from initial treatment, media were collected and analyzed by LDH assay. Values shown are a % of vehicle treatment 6 SD (B). doi:10.1371/journal.pone.0035566.g003 p,0.05) with the exception of the 10 mM YM-1 concentration (p.0.05). We attributed the lack of significance to the general toxicity caused by the 48-hour 10 mM concentration of YM-1 (see Figure 3A & B). A one-way ANOVA of the YM-1 concentration curve for the 4 hour YM-1 treatment followed by 44 hours of 4- OHT treatment revealed that the 10 mM concentration was significantly different from all other concentrations (F(4,10) = 16.49, p = 0.0002)(Figure 4C). The concentration curve for the 4 hour YM-1 treatment followed by 95% EtOH treatment displayed that no concentration was significant from any other concentration by one-way ANOVA (F(4,10) = 3.435, p = 0.0516)(Figure 4D). Comparison of all of the 48 hour YM- 1 concentrations, by one-way ANOVA, displayed that, again, the 10 mM concentration was significantly different from all other concentrations in this treatment (F(4,10) = 12.32, p = 0.0007)(Figure 4E). We continued our analysis by comparing the 10 mM YM-1 concentration, from all treatment groups, with the treatment. Viability values of all aforementioned Figure 2. YM-1 toxicity specific to cancer cells; non-cancer cells unaffected. MCF7 cells (breast cancer) were treated for 24 hours with increasing concentrations of YM-1. After 24 hours, medium was collected and analyzed by LDH cytotoxicity assay. Values shown are a % of vehicle treatment 6 SD (A). Hs578T and MDA-MB-231 (breast cancer), M17 (neuroblastoma), H4 (neuroglioma), and HeLa (cervical cancer) cell treated with increasing concentrations of YM-1 and the toxicity was compared to NIH-3T3 (mouse embryonic fibroblast) and HEK 293 (human embryonic kidney) cells for cancer specific toxicity. All cell lines were treated for 24 hours. After 24 hours, media were collected and analyzed by LDH assay. Values shown are a % of vehicle treatment 6 SD (B). doi:10.1371/journal.pone.0035566.g002 treatment and the 4-hour YM-1 followed by 95% EtOH treatment (all p.0.05); whereas, all the concentrations used for the 4-hour YM-1 followed by 4-OHT treatment were significantly different from the 4-hour YM-1 followed by 95% EtOH treatment (all p,0.05). All the concentrations of the 4-hour YM-1 followed by 4- OHT and the 48-hour YM-1 were significantly different (all PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35566