YM-1 were able to resensitize cancer cells that had developed resistance to the ERa antagonist, tamoxifen. One way in which these compounds are working is by reducing total Akt levels, which can contribute to ERa insensitivity to tamoxifen. Combined, the rhodacyanine scaffold holds great potential as a cancer therapeutic both as an individual treatment strategy but also, potentially, as a combinational or synergistic option for use with existing regimens. Methods Cell Lines Tamoxifen resistant (TR-MCF7) and parental MCF7 cells were generously provided by Dr. Jin Q. Cheng of Moffitt Cancer Center (Tampa, FL). The MCF7 line was originally generated by the Michigan Cancer Foundation and were obtained from ATCC (Manassas, VA) and the TR-resistance was produced by chronic low dose treatment with tamoxifen. HEK-293, M17, H4, MDA- MB-231, Hs578T and NIH-3T3 cells were purchased from ATCC (Manassas, VA). HeLa cells were generously provided by Dr. Kenneth E. Ugen at the University of South Florida. He originally obtained them from ATCC (Manassas, VA). These cells were generated from a cervical tumor from Henrietta Lacks. Chemicals and Antibodies Methylene blue (MB) was purchased from Sigma Aldrich (St. Louis, MO). MKT-077 and YM-1 were synthesized as described [19]. Anti-Akt1, Akt2, and pAktS473 were purchased from Cell Signaling Technology (Danvers, MA). Anti-ERa, and pERa S167 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Actin was purchased from Sigma Aldrich. Anti-GAPDH was purchased from Meridian Life Science (Memphis, TN). Cell Culture and Drug Treatments MCF7, MDA-MB-231, Hs578T and HeLa cells were grown as previously described [20]. H4 and HEK-293 cells were cultured in OPTI - modified Eagle’s medium (OPTI-MEM) from Invitrogen supplemented with 10% fetal bovine serum (FBS) and 1% PenStrep (Invitrogen). M17 cells were cultured in OPTI-MEM supplemented with 10% FBS, 1% PenStrep and 100 mg/L Sodium Pyruvate. NIH-3T3 cells were cultured in DMEM with low-sodium bicarbonate (1.5 g/L) from ATCC supplemented with 10% FBS and 1% PenStrep. TR-MCF7 cells were grown in DMEM (described with MCF7 cells) supplemented with 10 mM 4- OHT. MKT-077 and YM-1 were dissolved in DMSO. DMSO was used as vehicle for MKT-077 and YM-1 where indicated. Exact treatment strategies accompany data in results section. Protein Collection, Quantitation, and Western Blotting Cells were harvested by application of mammalian protein extraction reagent (Thermo) as previously described [20]. Protein level measurement, equilibration, western blotting, and detection were performed as previously described [20]. Lactate Dehydrogenase (LDH) Assay Indicated cell lines were plated in designated medium. Once cells reached ,95% confluency, MKT-077 or YM-1 was applied in DMEM without phenol red. After times indicated per experiment, medium was collected from each treatment and centrifuged to pellet dead cells and debris. Protocol was followed as supplied from Cytotox-96 kit (Promega). Rhodacyanine Derivative Kills Resistant Cancers Mitochondrial Isolation and Spectroscopy MCF7 cells were treated for 6 hours with vehicle (DMSO), MB, YM-1, or MKT-077. Following treatment cells were harvested and subcellular fractions collected using Pierce Mitochondrial Isolation Kit from Thermo Scientific (Rockford, IL). Analysis of drug localization was performed by spectroscopy on Thermo Scientific Nanodrop spectrophotometer. Concentrations and subsequent percentages were approximated by generated concen- tration:absorbance curve (not shown). MTT Cell Viability Assay TR-MCF7 cells were plated in a 96well plate in medium containing 10 mM 4-OHT. When cells reached ,90% confluency cells were treated in OPTI-MEM in one of four conditions 1: 10 mM 4-OHT in OPTI-MEM for the full 48 h of experiment. 2: YM-1 (or vehicle) at indicated concentrations for 4 hours followed by exchange of YM-1 medium with medium containing 10 mM 4- OHT. 3: YM-1 (or vehicle) at indicated concentrations for 4 hours followed by exchange of YM-1 medium with medium containing 95% EtOH (vehicle for 4-OHT). Or, 4: YM-1 (or vehicle) at indicated concentrations for the full 48 hours of experiment. MTT assay kit was purchased from ATCC and assay was run as per supplied protocol. Isolation of Nuclear Proteins TR-MCF7 cells were grown in designated medium in 10 cm dishes. Cells were treated for 4 h with 10 mM 4-OHT, 10 mM YM-1, both or vehicle(s) for both compounds. Following incubation, cells were harvested and nuclear proteins isolated using reagents and supplied protocol from the Qproteome Nuclear Protein Kit (Qiagen). Results The cytotoxicity profiles of a series of derivatives to MKT-077 on MCF7 cells were compared in a small-scale screen. The derivative YM-1 was the only compound found to have dose dependently higher toxicity than MKT-077 after 24 hours (LDH values normalized for cell number)(Figure 1A). One possible reason for this improved potency was cellular localization. Taking advantage of the unique spectral properties of these compounds, MCF7 cells were treated with MKT-077 or YM-1 and cellular separation of mitochondria and cytosol were performed. Methy- lene blue, a compound known to localize to the mitochondria, was used as a control [21]. The subcellular fractions were analyzed spectrophotometrically. These values were compared with a generated standard curve of Abs:concentration (data not shown) to give an approximate concentration of compound in each fraction and thus a percentage of drug per location. Interestingly, YM-1, unlike MKT-077 was more prevalent in the cytosolic fractions (Figure 1B). Concerned that the lack of mitochondrial interaction would reduce the cytotoxic specificity seen with rhodacyanine’s for cancer cells, the selectivity of YM-1 on several cancer and immortalized cells lines were tested. These included: MCF7, Hs578T and MDA-MB-231 (breast cancer), M17 (neuroblasto- ma), H4 (neuroglioma), HeLa (cervical cancer), and two immortalized cell lines: HEK 293 (human embryonic kidney) and NIH 3T3 (murine fibroblast). Robust cytotoxicity (1323% of vehicle), as measured by lactate dehydrogenase (LDH) assay, was observed in MCF7 cells following 24-hour YM-1 treatment (Figure 2A). As expected, these toxicity values were higher than those observed in Figure 1A since we used a larger cell population (1A<0.26106 cells; 2A<1.26106 cells) and thus more LDH was PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35566