transmembrane potential and induced apoptosis in MM cell lines. The combination of the anti IGF-1R antibody with AS602868 increased the cytotoxic effect in MM cell lines, suggesting that simultaneous targeting of IGF-1 signalling and the NF-kB pathway could be of therapeutic value in multiple myeloma. Results IGF-1R expression level In a first step, we measured the expression level of IGF-1Ra by flow cytometry in the different MM cell lines. We found that RPMI8226 and LP1 expressed more IGF-1Ra than MM.1S and U266 (Figure 1A). Since IGF-1 can also bind the insulin receptor (IR) and the insulin-like growth factor 2 receptor (IGF-2R), we analyzed the expression of IGF-1R, IGF- 2R and IR by quantitative RT-PCR. We confirmed that LP1 and RPMI8226 expressed a higher level of IGF receptor than MM.1S and U266 (Figure 1B). Combining the anti-IGF-1R monoclonal antibody with the IKK2 inhibitor AS602868 increases the apoptosis of MM cell lines In order to study the effect of the combination of the IKK2 inhibitor AS602868 and the anti- IGF-1R monoclonal antibody on four human MM cell lines, we incubated cells with various concentrations of AS602868 alone or in combination with 10 mg/ mL anti-IGF-1R. While AS602868 was cytotoxic against the four cell lines studied, the combination with anti-IGF-1R antibody enhanced cytotoxicity significantly only in RPMI8226 and LP1 cells (Figure 2A). IC50 for AS602868 were reduced from 35 mM to 10 mM upon addition of anti-IGF-1R in RPMI8226 cell line and 39 mM to 7 mM in LP1 cell line. The combination of anti-IGF-1R with AS602868 had not any additional effect on MM.1S and U266 cell lines (Figure 2A) which express lower levels of IGF-1R. Same experience is performed without serum; such as the first experience, the effect effect of AS602868 was enhanced by anti- IGF-1R antibody (Figure S2). To determine if the potential effect of anti-IGF-1R was related to the inhibition of IKK2, we studied the viability and apoptosis of Anti-IGF-1R Potentiate the Effect of AS602868 MM cell lines in presence of the selective IKK2 inhibitor, TPCA- 1. This inhibitor decreased the viability of the four cell lines. However, the cytotoxic effect of TPCA-1 was enhanced by anti- IGF-1R only in LP1 and RPMI8226 cells (Figure 2B). To confirm these results, we measured the induction of apoptosis by annexin V staining in cells exposed to 10 mM AS602868, TPCA or/and 10 mg/mL anti-IGF-1R antibody. Interestingly, the four cell lines showed an apoptotic response upon incubation for 72 hours with 10 mM AS602868, TPCA-1 or/and 10 mg/mL anti-IGF-1R. However, anti-IGF-1R antibody enhanced apoptosis induced by these agents only in the in LP1 and RPMI8226 cell lines (Figure 2C). Growth inhibition studies showed that MM.1S and U266 were more resistant to anti-IGF-1 then LP1 and RPMI8226 (Figure S1). The combination of anti-IGF- 1R with AS602868 or with TPCA-1 was found to increase the apoptotic fraction in RPMI8226 and LP1 cells but not in MM.1S and U266 cells (Figure 2C). To understand how the inhibition of IKK2 and the IGF-1R antibody could cooperate to cause cell death in myeloma cells we then chose to focus on the two cells lines with the highest levels of IGF-1R, LP1 and RPMI8226. Combining the anti-IGF-1R monoclonal antibody with the IKK2 inhibitor AS602868 increases the apoptosis of primary myeloma cells Since anti-IGF-1R antibody enhanced the cytotoxicity effect of the IKK2 inhibitors on MM cell lines, we evaluated the effect of this combination on primary MM cells. We incubated primary myeloma cells with IKK2 inhibitors or/and anti-IGF-1R antibody for 24 h. We analyzed the effect of these signalling pathways inhibitors on CD221+ and CD2212 plasma cells. About 30% of primary plasma cells express IGF-1R (CD221+) (Table S3). After 24 h, anti-IGF- 1R antibody increased the apoptotic fraction from 5% to 20% in CD221+ plasma cells with no significant effect on CD2212 plasma cells (Figure 2D). The effect of AS602868 was more pronounced on CD221+ plasma cells then on CD2212 plasma cells. The apoptotic effect of the combination was detected only on CD221+ plasma cells (Figure 2D). Anti-IGF-1R antibody and AS602868 were not any cytotoxicity effect on bone marrow non plasma cell compartment (Table S4). Figure 1. Level expression of IGFs receptors on four MM cell lines. 1.A Level expression of IGF-1Ra by flow ctometry on RPMI8226, LP1, MM.1S and U266. 1.B Expression of IGF-1R, IR and IGF-2R by RT-PCR on RPMI8226, LP1, MM.1S and U266. doi:10.1371/journal.pone.0022641.g001 PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e22641