Anti-IGF-1R Potentiate the Effect of AS602868 Figure 3. AS602868 and anti-IGF-1R antibody block the cell cycle in RPMI8226 and LP cell lines. 3.A Effect of AS602868 and anti-IGF-1R antibody on the cell cycle. MM cell lines were exposed for 40 h to 10 mM AS602868 or/and 10 mg/mL anti-IGF-1R antibody at 37uC. Cells were incubated with propidium iodide and then analyzed with flow cytometry. 3.B Analysis of cell cycle proteins in RPMI8226 cells exposed to AS602868 or/and anti- IGF-1R. RPMI8226 cells were incubated with 10 mM AS602868 or/and anti-IGF-1R antibody 10 mg/mL for 16 h then examined by Western blotting. Mean6 SD of three experiments ** P,0.001: * P,0.05 versus control. doi:10.1371/journal.pone.0022641.g003 level of cytochrome c by western blot by flow cytometry in RPMI8226 and LP1 cells. We detected an increase of cytochrome c protein content in cells incubated with AS602868. While anti IGF-1R antibody had not effect on cytochrome c content its association with AS602868 clearly increased the increase of cytochrome c, in particular in LP1 cells which are those with the highest level of expression of IGF-1R (Figure 4B). Effect of AS602868 and anti-IGF-1R antibody on NF-kB signalling During activation, IKK2 is phosphorylated and induces the phosphorylation of IkBa. In order to study the effect of the combination anti-IGF-1R and the IKK2 inhibitor on NF-kB, we analysed the phosphorylation status of IKK2 as well as that of its substrate IkBa by western blot. As expected incubation of RPMI8226 with AS602868 for 16 h decreased the phosphoryla- tion of IKK2 with a resulting decrease in phospho-IkBa. AS602868 also caused a decrease in p65 but no change in phospho-p65. Conversely anti-IGF-1R antibody decreased the phosphorylation of both IkBa and p65, suggesting that blockage of IGF-1 signalling has a profound inhibitory effect on NF-kB signalling (Figure 5A). Remarkably the reduction of phospho-IkBa was maximal when anti-IGF-1R antibody was combined to AS602868. These results suggest that inhibition of IGF-1 signalling potentiates the repression of NF-kB by an IKK inhibitor such as AS602868. The ability of the combination of anti-IGF-1R and an IKK2 inhibitor to interfere with NF-kB activation was also analyzed by RT-PCR. Generally, the activation of NF-kB is accompanied with an increased mRNA content of various molecules such as IL-6 and Inter-Cellular Adhesion Molecule 1 (ICAM-1). In RPMI cell lines, we observed that anti-IGF-1R does not have a significant effect on IKB, IGF-1, IL-6 and ICAM-1 (Figure 5B). While the IKK2 inhibitor decreased the expression level of IkB, IL-6 and ICAM-1 (Figure 5B), this effect was more pronounced, both in LP1 and RPMI8226 cells, when anti-IGF-1R antibody was combined with AS602868. Finally, we studied by RT-PCR the level of IGF-1. Interestingly, we found that the combination suppressed the level of IGF-1 whereas anti-IGF-1R or AS602868 alone did not have any effect on IGF-1 mRNA content in these cells (Figure 5B). Level expression of IGF-1 protein was detected also by western blot. The level expression of protein IGF-1 decreases in cell exposed to the combination anti-IGF-1R and AS602868 (Figure S3). Effect of AS602868 and anti-IGF-1R antibody on PI-3K/Akt pathways Since IGF-1 is involved in the activation of PI-3K/Akt pathways, we analysed the effect of its inhibition on the RPMI8226 cell line. We observed that the phosphorylation of Akt decreased in cells exposed to anti-IGF-1R. This inhibition of Akt phosphorylation causes a subsequent inhibition of the phosphorylation of the mTor pathway proteins, p70S6K and 4EBP1 (Figure 6A). Effect of AS602868 and anti-IGF-1R antibody on Bcl-2 family proteins As anti-IGF-1R-induced apoptosis of MM cell lines was not associated with depolarization of the mitochondrial membrane, we sought to determine whether alteration in IGF-1 signalling could alter the content of pro-apoptotic and anti-apoptotic proteins. We observed that anti- IGF-1R antibody increased the content of pro-apoptotic proteins Bim and Bad and decreased those of anti-apoptotic proteins Bcl-2 and Bcl-xL (Figure 6B). AS602868 did not affect the content of these proteins under these conditions. PLoS ONE | www.plosone.org 4 July 2011 | Volume 6 | Issue 7 | e22641