9.2.27PE and ABT-737 in Melanoma Figure 3. 9.2.27PE in combination with ABT-737 induces strong depolarization of the mitochondrial membrane. (A) FEMX and Melmet-5 cells were treated with 9.2.27PE (100 ng/ml)6ABT-737 (10 mM) for 24 h and subsequently stained with JC-1, an indicator for mitochondrial membrane potential. A decrease in the red/green fluorescence ratio indicates decreased mitochondrial membrane potential. CCCP was used as a depolarization control. The figures shown are examples from one representative experiment. The red/green ratio value is based on three independent experiments. (B) DNA fragmentation was analyzed by TUNEL assay. FEMX and Melmet-5 cells were treated with 9.2.27PE (100 ng/ ml)6ABT-737 (10 mM) for 24 h. In addition, the Melmet-5 cells were pre-treated with for 1 h with Z-VAD-FMK (50 mM)6Z-FA-FMK (50 mM) to determine whether caspases and cathepsins were involved in the DNA fragmentation observed in this cell line. doi:10.1371/journal.pone.0024012.g003 9.2.27PE and ABT-737 reduces tumor growth in vivo Our in vitro data demonstrated that a combination of 9.2.27PE and ABT-737 caused synergistic cytotoxic effects in a panel of melanoma cells. To determine whether these effects could be validated in vivo, nude mice with subcutaneous Melmet-5 xenografts were treated with 9.2.27PE and ABT-737. As seen in Figure 6A, the combination regime of 9.2.27PE (31.25 mg/ kg)+ABT-737 (100 mg/kg) caused significant cytostatic effect after 21 and 25 d (p,0.05). As all animals receiving 100 mg/kg ABT- 737 developed skin rash, the concentration of ABT-737 was halved in the second experiment (Fig. 6B). Skin rash was not observed, but the combination treatment did not significantly decrease inhibition of tumor growth, possibly due to large tumor volume variations within the groups. There was, however, a tendency to stronger cytostatic effect caused by the combination treatment compared to treatment with ABT-737 or 9.2.27PE as single agents. Body weight was stable throughout the experiments (Figure S4). Discussion Cancer cells are characterized by uncontrolled proliferation and apoptotic resistance. Melanoma cells are particularly drug resistant, and drug-resistance is often correlated with apoptosis evasion [2]. Drug resistance is likely a reason for treatment failure PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e24012