been related to proliferation and drug resistance of MM cells, confirming the importance of inhibing NF-kB as a therapeutic target in MM [5]. MM cells have been shown to be sensitive to NF-kB inhibitors including proteasome inhibitors and IKK inhibitors [22,49,50]. Preclinical and clinical studies have shown that the IKK2 inhibitors AS602868 and TPCA-1 induce apoptosis in MM cells by decreasing the canonical NF-kB pathway [27]. In our study, we observed the effect of the combination of monoclonal anti-IGF-1R antibody and IKK2 inhibitors. Interestingly, among the four cell lines with different expression levels of IGF-1R which we studied, only in those with the highest levels did we observe enhanced cytotoxic activity of IKK2 inhibitors by anti-IGF-1R antibody. We observed that the apoptotic response of MM cells to AS602868 involved disruption of the mitochondrial transmembrane potential and release of cytochrome c. While anti-IGF-1R antibody did not induce mitochondrial membrane depolymerisation it did alter the expression levels of pro- and anti-apoptotic proteins, possibly through inhibition of PI3K signalling, a factor which might explain why the combination with AS602868 resulted in greater cytochrome c content than that caused by AS602868 alone. Moreover, inhibition of IGF-1 signalling by the anti-IGF-1R antibody, while in itself did not reduce NF-kB signalling, resulted in decreased phospho-IkB, thereby setting the stage for the effect of AS602868 on NF-kB signalling. The inhibition of NF-kB was associated with a reduction in the expression of adhesion molecule ICAM-1 and IL-6. In this study, we show that the combination of two pathways inhibitors decreased the production of IGF-1 and IL-6. Altogether, our data underscore the central role for IGF-1 in MM and the importance of IGF-1 signalling as a potential therapeutic target. Interestingly our results suggest that inhibition of IGF-1 signalling in MM could be used as a chemosensitization strategy rather than as a cytotoxic approach per se. We indeed observed that exposure to anti-IGF-1R antibody contributed to mitochondrial membrane disruption and NF-kB inhibition caused by the IKK inhibitor AS602868. These results were all the more remarkable as these two agents had different effects on the cell cycle and that the combination of the two had an effect super imposable to that of the anti-IGF-1R antibody. These findings suggest that a strategy combining monoclonal antibody against IGF-1R with NF-kB inhibitors may constitute a promising therapeutic approach in patients with MM. Materials and Methods Cell lines Human multiple myeloma cell lines LP1, RPMI8226, U266 and MM.1S were cultured in RPMI 1640 containing L-Glutamine (Invitrogen, Cergy Pontoise, France), 10% fetal bovine serum (Invitrogen, Cergy Pontoise, France) and 1% penicillin strepto- mycin (Invitrogen, Cergy Pontoise, France) at 37uC in humidified 95% air and 5% CO2. LP1, MM.1S and U266 were provided by ATCC-LGC (Molsheim, France). RPMI8226 was provided by ECACC from sigma-Aldrich (Sigma Aldrich, St Louis, USA). Growth inhibition Assay The cell survival was assessed by using a 3-(4,5-dimethylthiazol- 2-yl)-2,5- diphenyltetrazolium bromide assay (MTT, Sigma Aldrich, St Louis, USA). Cells were cultured in 96-well plates (Costar, NY, USA) for 72 h in the presence of AS602868 (generously provided by Merck-Serono, Geneva, Switzerland), TPCA-1(Sigma Aldrich, St Louis, USA) alone or with an anti- IGF-1Rmonoclonal antibody (generously provided by Roche, Penzberg, Germany). Cells were then incubated with 100 mg Anti-IGF-1R Potentiate the Effect of AS602868 MTT, and the resulting crystals were resuspended in 100 mL of isopropanol/0.1 N HCl (Sigma Aldrich, St Louis, USA). Absor- bance was measured at 540–690 nm using a spectrophotometer (Thermo Electron Corporation), and inhibitory concentrations 50 (IC50) were determined from cell survival curves drawn with Excel (Microsoft). Fresh human myeloma cell studies We received 2 mL samples of bone marrow (BM) from patients with multiple myeloma, after having obtained written informed consent. Samples were lysed with lysis buffer for 10 minutes at room temperature. BM cells were washed and suspended in 300 ml phosphate buffer (PBS). Cells were cultured in 24 well plates (Costar, NY, USA) at 37uC with AS602868 (10 mM), anti-IGF-1R (10 mg/mL) or AS602868+anti-IGF-1R. After 24 h, cells were washed with PBS for 5 minutes, centrifuged at 600 g at room temperature and incubated with labelled monoclonal antibodies directed against CD 38, CD 138, CD221(IGF-1R alpha) and CD45 (antibodies Reference in Table S2) for 15 minutes in the dark at room temperature. Cells were washed with PBS and suspended in 100 mL Annexin buffer with 1 ml Annexin-V FITC and analysed by flow cytometry on a FACS Canto II (BD Biosciences, Erembodegem, Belgium). Western blot analysis Cells (1.107) were exposed to 10 mM AS602868 or/and 10 mg/ mL anti-IGF-1R for 16 hours. Following treatment, cells were washed with phosphate-buffered saline (PBS) and then solubilised for 30 minutes on ice in lysis buffer (20 mmol/L Tris-Hcl (pH 6.8), 1 mmol/L MgCl2, 2 mmol/L EGTA, 0.5% NP40) with the complete mixture of protease inhibitors (leupeptin, aprotinin, benzamidine, PMSF, TRCK). After incubation, cell debris and nuclei were removed by centrifugation at 12,000 g for 15 minutes at 4uC. Equal amounts of total protein (50 mg per lane) combined with Laemmli buffer were boiled at 95uC for 5 minutes, and then separated on 12% sodium dodecyl sulphate (SDS)- polycrylamide gels followed by electrophoretic transfer to iBlot Gel transfer stacks PVDF or nitrocellulose (Invitrogen). Membranes were blocked for 1 hour at room temperature with phosphate- buffered saline containing 0.1% TweenH 20 (PBS-T) and 5% dried milk and incubated for all night at 4uC with specific antibody diluted with PBS-T and 5% non fat milk. The antibodies used were directed against Bcl-2 (M0887, 1/500, Dako, Denmark), Bcl- XL (sc-634,1/500, Santa cruz, USA), IGF-1 (sc-1422, 1/500, Santa cruz, USA), P21(sc-397, 1/500, Santa cruz, USA), P53 (clone DO7, 1/1000, Dako, Denmark), IkBa(ser32/ser36, IMG- 156,1/500 IMGENEX, USA), Bim (2819), Bad (610392, 1/250, BD transduction, USA), IKKb (2684), IKKb (2681), phospho- IKBa (S32/S36) (9426), NF-kB p65 (C22B4), phospho-NF-kB p65 (Ser536), 4EBP 1(9644), Phospho 4EBP-1(2855), p70S6k (2708), phospho p70S6k (9209), cyclin E (4129), cyclin A (4656) (1/1000 or 1/2000, Cell Signalling, USA), Akt (IMG-5411-1, 1/250, Imgenex, USA), phospho Akt (IMG-187-A, 1/500, Imgenex, USA)). Membranes were washed with PBS-T 5% dried milk and incubated with a secondary antibody against mice or rabbit immunoglobulin (1/3000, Caltag, USA) for 1 h. Signal was detected by chemoluminescence using ECL western blotting detection system (GE Healthcare, UK). b- actin clone AC-15 (Sigma Aldrich, St Louis, USA) served as internal control for equal loading. Cell cycle analysis Cellular DNA content was determined by flow cytometry. After incubation with 10 mg/mL anti-IGF-1R and/or 10 mM PLoS ONE | www.plosone.org 7 July 2011 | Volume 6 | Issue 7 | e22641