9.2.27PE and ABT-737 in Melanoma Figure 6. Effect of 9.2.27PE in combination with ABT-737 in a Melmet-5 xenograft model. (A) Nude mice were treated with ABT-737 (100 mg/kg) day 1–5 and 15–19 and 9.2.27PE (31.25 g/kg) on day 1 and 15. (B) Nude mice were treated with ABT-737 (50 mg/kg) day 1–5 and 15–19 and 9.2.27PE (31.25 g/kg) on day 1 and 15. Tumor size was measured 1–3 times weakly by caliper measurements and tumor volume was calculated (tumor volume = L6W2/2). * indicates p value,0.05, using ANOVA. Arrows indicate the day animals in the combination group were sacrificed due to adverse events (skin ulcers). doi:10.1371/journal.pone.0024012.g006 system, hence, these proteins may serve as an important target for anti-cancer immunotherapeutic strategies [28]. The combined use of several drugs targeting different cellular functions, e.g. the Bcl-2 family, is therefore one approach to achieve tumor control in cancer, including melanoma. In the present study we show that the melanoma specific immunotoxin 9.2.27PE in combination with the BH-3 mimetic ABT-737, caused strong synergistic cytotoxic effect in the panel of melanoma cells, independently of their BRAF status and, as previously reported [20], their sensitivity to Dacarbazine. Depolarisation of the mitochondrial membrane, activation of caspase-3, inactivation of PARP and increased DNA fragmenta- tion was obtained using the combination treatment, hence, cell death involved induction of the intrinsic apoptotic pathway. To elucidate which mechanisms are responsible for the synergistic cytotoxic effect observed when treating melanoma cells with 9.2.27PE+ABT-737, we looked to the ER, as ABT-737 has been reported to induce the transcription factor ATF4 in other types of cancer [29,30]. Disturbances in the normal functions of the ER lead to unfolded protein response and ER stress [31]. The initial intent of the unfolded protein response is to adapt to these changes and re-establish normal ER function. This happens through dissociation of GRP78 from the three ER chaperones PERK, IRE1 and ATF6, resulting in increased protein levels of GRP78 and activation of the three ER stress pathways (PERK– eIF2a–ATF4, ATF6-ATF6 splicing, and IRE1-XBP1) [31,32]. Unresolved and prolonged ER stress will eventually result in apoptosis [32]. In this study, ABT-737 treatment resulted in increased protein levels of GRP78 and peIF2a, indicative of ER stress. In addition, ABT-737 treatment resulted in decreased Dym, inactivation of PARP and increased sub-G1 population, mecha- nisms related to apoptosis. These results indicate that ABT-737 as a single agent induces ER stress which ultimately results in cell death. Another function of the ER is storage and regulation of calcium. Upon ER stress, calcium is released from the ER into the cytosol which can be taken up by juxtaposed mitochondria and result in decreased Dym [33]. In this study we show that ABT-737 increased [Ca2+]i levels, and that 9.2.27PE, which as a single agent had minimal effect on calcium release, enhanced the ABT-737 induced calcium release from ER. This result suggests that 9.2.27PE directly or indirectly has an effect on ER and is effective for sensitizing the melanoma cells to ABT-737. Others have shown that decreased Mcl-1 expression is effective for sensitizing cancer cells to ABT-737 and induce apoptosis [15,16]. As 9.2.27PE lead to rapid decrease in Mcl-1 protein levels [11], this lead us to investigate whether knockdown of Mcl-1 protein expression could increase the [Ca2+]i levels, decrease the Dym and decrease the cell viability in ABT-737 treated melanoma cells. In contrast to the control cells (MelRM and MelRMshCtr), ABT-737 treated MelRMshMcl-1 cells showed significantly increased [Ca2+]i levels, decreased Dym and decreased cell viability, effects which could not be further enhanced by 9.2.27PE in the MelRMshMcl-1 cells. These results indicate that Mcl-1 has an inhibitory effect on calcium release from the ER, and that strongly decreased Mcl-1 protein levels in melanoma cells after treatment with 9.2.27PE (Fig. 4A) might be responsible for enhanced [Ca2+]i levels in ABT- 737 treated cells. Calcium release has been shown to take place through the inositol 1,4,5-triphosphate receptor and the ryanodine receptors in the ER membrane [13,31]. Bcl-2 family proteins are involved in this process, as several members, including Mcl-1, have been shown to interact with e.g. inositol 1,4,5-triphosphate receptor [34–38]. The precise mechanisms how the 9.2.27PE+ABT-737 drug combination causes calcium release and synergistic cell death in melanoma cells is under investigation. The downstream effectors of increased [Ca2+]i levels, are numerous and thoroughly reviewed by Xu et al [31]. Of importance to the present study is the mitochondrial uptake of excessive Ca2+ leading to decreased Dym, activation of the intrinsic apoptotic pathway via release of a number of pro- apoptotic factors including cytochrome c, apoptosis inducing factor (AIF) and Smac/Diablo [39]. The enhanced [Ca2+]i levels caused by 9.2.27PE+ABT-737 treatment could therefore explain the increased mitochondrial membrane depolarization caused by the PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e24012