9.2.27PE and ABT-737 in Melanoma Figure 1. 9.2.27PE in combination with ABT-737 causes synergistic cell cytotoxicity in melanoma cells. (A) The 9.2.27PE immunotoxin caused a time- and dose dependent decrease of cell viability in melanoma cells. The effect of the immunotoxin was similar in the different cell lines tested. MM200: 24 h experiment only. (B) ABT-737 caused decreased cell viability in a panel of melanoma cells. The cell cytotoxic effect of ABT-737 differed between the cell lines. (C) B-RAF status and IC50 values of ABT-737 treated melanoma cells. WT = wild type, M = BRAFV600E, UNK = unknown. (D) The CalcuSyn software program was used to calculate synergistic, additive or antagonistic effects. Combining 10 ng/ml 9.2.27PE+1 mM ABT-737 or 100 ng/ml 9.2.27PE+10 mM ABT-737 caused combination index (CI) values in the 0.59 to 0.003 range indicating synergy to very strong synergy after 24–48 h. doi:10.1371/journal.pone.0024012.g001 (100 ng/ml)+ABT-737 (10 mM) and 9.2.27PE (10 ng/ml)+ABT- 737 (1 mM) in FEMX cells (Fig. 2D and data not shown), combinations which caused synergistic cytotoxic effects (Fig. 1D). A caspase-3/7 activity assay was utilized to confirm activity of the cleaved caspase-3 band detected on Western Blot. As seen in Figure S2, 9.2.27PE6ABT-737 increased caspase-3/7 activity after 24 h in both the FEMX and the Melmet-5 cells. The caspase- 3/7 acticity was effectively blocked by pre-treatment with the caspase-3 II inhibitor (Z-DEVD-FMK, 50 mM). STS (1 mM), which we previously have shown to induce caspase-3/7 activity in FEMX cells [11], was used as a positive control, and CHX, not causing caspase-3/7 activity [25], was used as a negative control. 9.2.27PE in combination with ABT-737 cause strong depolarization of the mitochondrial membrane Decreased Dym is linked to apoptosis as pro-apoptotic proteins, which are released from the mitochondria into the cytosol, are capable of activating caspase-3 and inducing DNA fragmentation. We used the cation dye JC-1 to detect whether the Dym was affected by 9.2.27PE6ABT-737 in FEMX and Melmet-5 cells. JC-1 fluoresces red in intact mitochondria, and upon collapse of the membrane, JC-1 accumulates in the cytoplasm where it fluoresces green. A decrease in red/green ratio indicates depolarization of the mitochondrial membrane. In FEMX cells, the red/green ratio decreased from 0.62 and 0.52 for the 9.2.27PE and ABT-737 treatments, respectively, to 0.22 for the combination treatment (Fig. 3A). Similar results were obtained for the Melmet-5 cells, where the red/green ratio decreased from 0.77 and 0.90 for the 9.2.27PE and ABT-737 treatments, respectively, to 0.15 for the 9.2.27PE+ABT-737 combination. CCCP was used as a positive control for depolarization. We next analysed DNA fragmentation by TUNEL assay. As seen in Figure 3B, 24 h treatment with 9.2.27PE and ABT-737 caused DNA fragmentation in ,20% and 5% of the Melmet-5 cells, respectively. Combining the two drugs, the percentage of DNA fragmentation increased to ,60%. Pre-incubation with Z- VAD-FMK+Z-FA-FMK inhibited the DNA fragmentation, indi- cating that caspases and cathepsins are involved in the cell death process. For the combination treatment, the caspase-3/7 activa- tion in Melmet-5 cells (Figure S2) did not directly correlate with the strong increase in DNA fragmentation (Fig. 3B) suggesting that other factors than caspase-3/7 might be involved in DNA fragmentation [26]. In a previous study with 9.2.27PE treated FEMX cells, no DNA fragmentation was detected using the TUNEL assay [11]. In the present study, no DNA fragmentation was detected in FEMX cells treated with 9.2.27PE6ABT-737 (Fig. 3B). As the combination treatment caused strong depolar- ization of the mitochondrial membrane in addition to caspase-3 activity, we conclude that under the above mentioned conditions, and after treatment with STS [11], DNA fragmentation does not take place in the FEMX cells. ABT-737 induces ER stress and cell death We wanted to investigate which mechanisms could contribute to the synergistic cell death effect observed by the 9.2.27PE+ABT-737 combination treatment. As prolonged ER stress can lead to apoptosis and autophagy [13], we first investigated whether ABT- 737 was able to induce ER stress in melanoma cells. The commonly used markers for ER stress, GRP78 and peIF2a, were increased in the ABT-737 treated FEMX, Melmet-5 and MelRM cells (Fig. 4A), the increase was more pronounced in the Melmet-5 and MelRM cells. Treatment with 9.2.27PE did not induce increased protein levels of GRP78, however an increase in peIF2a was observed. Tunicamycin, which is known to induce ER stress, was used as a positive control for increased GRP78. During ER stress, GRP78 dissociates from the three ER membrane proteins PERK, IRE1 and ATF6. This process leads to activation of these proteins, transcriptional activation of CHOP and may result in apoptosis [27]. CHOP was induced upon 9.2.27PE treatment, but interest- ingly not upon ABT-737 treatment in FEMX, Melmet-5 or MelRM cells despite increased levels of GRP78 and peIF2a. Further investigations are needed to clarify the involvement of CHOP in 9.2.27PE and ABT-737 treated melanoma cells. Nevertheless, ABT-737 treatment caused decreased Dym (Fig. 4B), inactivation of PARP (Fig. 4A) and increased sub-G1 fraction (Fig. 4C), after 48 h in FEMX, Melmet-5 and MelRM cells. These results indicate that ABT-737 was able to induce ER stress and cell death in melanoma cells in vitro. 9.2.27PE enhances Ca2+ efflux from Endoplasmic reticulum induced by ABT-737 One function of the ER is storage and regulation of calcium. We therefore investigated whether 9.2.27PE6ABT-737 caused calcium release from the ER in melanoma cells. ABT-737 caused an increase in intracellular calcium ([Ca2+]i) levels in FEMX, Melmet-5 and MelRM cells after 16 and 24 h (Fig. 5A). The increased [Ca2+]i levels was most pronounced in FEMX cells. The 9.2.27PE, which as a single agent had minor effect on calcium release, enhanced the [Ca2+]i levels caused by ABT-737. The enantiomer A-793844 was not able to elicit increased [Ca2+]i levels in Melmet-5 or MelRM cells after 16 and 24 h (data not shown). FEMX cells were not tested with the A-723844 for this purpose. In contrast to the control cells (MelRM and MelRMshCtr), silencing of Mcl-1 in the MelRM (MelRMshMcl-1) lead to significantly increased [Ca2+]i levels, decreased Dym and de- creased cell viability after 12 h ABT-737 treatment, effects which could not be further enhanced by 9.2.27PE (Fig. 5B). As the Mcl-1 level after 12 h 9.2.27PE treatment was undetectable (Fig. 5B) in all three cell lines, these results indicate that silencing of Mcl-1 through shRNA or 9.2.27PE treatment is responsible for the increased [Ca2+]i levels during ABT-737 treatment. The control cells, MelRM and MelRMshCtr, showed only a significant increase in [Ca2+]i levels when treated with 9.2.27PE+ABT-737, ant not with ABT-737 as a single agent treatment. The 9.2.27PE had no effect on calcium release in either of the MelRM cell lines after 12 h. Interestingly, 9.2.27PE caused less decrease of cell viability in MelRMshMcl-1 cells compared to control cells after 12 h (Fig. 5B) and 24 h (Figure S3), indicating a role of Mcl-1 in mediating the cytotoxicity of 9.2.27PE. PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24012