Anti-IGF-1R Potentiate the Effect of AS602868 Figure 4. AS602868 induces the depolarization of mitochondrial membrane. 4.A AS602868 and anti-IGF-1R antibody increased the depolarization of mitochondrial membrane in RPMI8226 and LP1 cell lines. Cells were incubated with 10 mM AS602868 or/and anti-IGF-1R antibody 10 mg/mL for 16 h then analysed by flow cytometry. 4.B AS602868 increased the level expression of cytochrome c in RPMI8226 and LP1 cell lines. Cells were incubated with 10 mM AS602868 or/and anti-IGF-1R antibody 10 mg/mL for 16 h. doi:10.1371/journal.pone.0022641.g004 Discussion Despite the fact that several treatments for multiple myeloma are currently available, the median survival of multiple myeloma remains in the order of 5 years, in particular in patients who are not eligible for high dose therapy. Many studies proved the importance of the activation of signalling pathways in chemo- resistance, cell survival, and proliferation of human multiple cells lines [30]. This makes these signalling pathways a therapeutic target in multiple myeloma. Over the past few years, several studies have shown that inhibition of these signalling pathways by specific inhibitors or by blocking the activator of these pathways causes an inhibition of cell proliferation [31,32]. The prolifer- ation and the survival of MM cells are strongly dependent on the activation of signalization pathways by cytokines and growth factors [33,34,35]. Most of these cytokines and growth factors are secreted by the microenvironment, in particular the bone marrow stromal cells [36,37]. IGF-1 is now considered as a major growth factor in MM [38]. Previous studies identified a role of IGF-1 as a growth and survival factor in MM. In vivo and in vitro studies proved IGF-1 increase antiapoptotic proteins (such as Bcl-2, Bcl-XL, cIAP-1, cIAP-2, FLIP) and decrease pro apoptotic proteins (such as caspase 3, caspase 8, caspase 9) and plays a role in drug resistance (dexamethasone, rapamy- cine)[39,40,41] Many studies in multiple myeloma have shown that the role of IGF-1 is correlated with signalling pathway activation. IGF-1 plays a major role in NF-kB, PI-3K/Akt and ras/MaPK activation [12,30,42].The inhibition of the interac- tion between IGF-1 and its receptor is being explored as a therapeutic target in this disease [43,44,45,46]. In vitro and in vivo studies have proved that the inhibition of of IGF-1R decreased cell proliferation [16,44,47]. Our results confirm the wide- ranging effect of IGF-1 inhibition on myeloma cells, including blockage of the G1 to S phase, reduced PI3K signalling and altered equilibrium of pro- and anti-apoptotic proteins. We show that the cytotoxic effect of anti-IGF-1R is more important on MM cell lines with a high level of IGF- 1R. In primary MM cell lines, anti-IGF-1R antibody enhanced the apoptotic effect of the IKK2 inhibitor AS602868 only in plasma cells with high expression of IGF-1R. Constitutive nuclear NF-kB activity has been described in many MM cells lines and primary myeloma cells [48]. Spontaneous and abnormal activation of NF-kB has PLoS ONE | www.plosone.org 5 July 2011 | Volume 6 | Issue 7 | e22641