Insulin-Like Growth Factor 1 Potentiates the Cytotoxic Activity of Bortezomib Against Myeloma Cells

Abstract 3987

Multiple Myeloma (MM) is a clonal plasma cell disorder whose growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type 1 (IGF-1). Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma and has been suggested to induce apoptotsis by reducing NF-κB signalling. Other cytotoxic mechanisms have been suggested, including increased reticulum stress leading to an unfolded protein response. We analyzed the impact of recombinant IGF-1 combined with the proteasome inhibitor bortezomib on human plasma cell lines in vitro and in vivo and on fresh human myeloma cells ex vivo. Using an MTT assay, we found that IGF-1 enhanced the cytotoxic activity of bortezomib in vitro against the LP1, RPMI8226, U266 and MM1.S lines, at a concentration of IGF-1 of 100 ng/mL. This potentiating effect was confirmed on MM1.S cells using a flow cytometric analysis of annexin V staining, and showed that the enhanced toxicity could be inhibited by the presence of a monoclonal antibody directed against the IGF-1 receptor (IGF1-R). IGF-1 was also found to enhance the cytotoxic activity of other proteasome inhibitors against MM1.S cells, including MG115, MG132, PSI and epoximicin. In vivo studies were performed in SCID mice bearing MM1.S xenografts. Mice received weekly administrations of bortezomib (0.5 mg/kg, i.p.) with or without recombinant IGF-1 (0.03 mg/kg, i.p.). The co-administration of IGF-1 with bortezomib significantly delayed tumor growth in comparison to that observed in mice treated with bortezomib alone. Fresh human myeloma cells exposed to bortezomib ex vivo displayed a larger annexin V positive fraction when they were co-incubated with IGF-1 then when they were exposed to bortezomib alone. This effect, which could be observed in subpopulations of CD45 hi and CD45 lo cells, could be reversed by an antibody directed against IGF-1R. Thus in each of these situations, IGF-1 increased the sensitivity of multiple myeloma cells to the cytotoxic effect of bortezomib. Analysis of pro- and anti-apoptotic proteins in MM1.S cells by immunoblotting showed that the addition of IGF-1 to bortezomib significantly enhanced the content o Bax, Bad and Bak and significantly reduced the content of Bcl2, BclX-L and Bfl-1. Exploration the NFkB pathway showed that exposure to IGF-1 and bortezomib induced a reduction of IkBalpha, an increase in phosphor-IKBalpha as well as a decrease in NFkB p65. Other observations made with the IGF-1/bortezomib combination include an increase in the content of cleaved caspase 3 and in P21 protein. Cell cycle distributions of cells exposed to bortezomib alone or the IGF-1/bortezomib combination were similar. Preliminary data showed an increased content of CHOP protein, suggesting that the IGF-1/bortezomib combination might enhance reticulum stress in MM1.S cells, thus leading to an Unfolded Protein Response (UPR) and to cell death. These results suggest that IGF-1 sensitizes myeloma cells to proteasome inhibitors by contributing to the enhancement of the reticulum stress. Overall these results suggest that exposure of myeloma cells to one of their key growth factors, IGF-1, significantly enhanced their sensitivity to bortezomib as well as to other proteasome inhibitors. This phenomenon appears to involve several pathways and may be dependent on the high baseline level of reticulum stress present in myeloma cells.