EM inhibited the serumstimulated proliferation of MCF and NCIH cell lines over many serum concentrations tested. We then explored the question if cells that were first stimulated with IGFI could still be inhibited in their proliferation and survival by a later incubation with EM.MCF cells were first activated with IGFI for min before EM was added, and the cell viability was measured after days by MTT assay. The inhibition by EM was similar to that observed when the cells were first incubated with EM for min and then IGFI was added. In a separate experiment, the simultaneous addition of IGFI and EM also resulted in a potent inhibition, which was similar to that obtained upon an initial incubation with EM for hbefore the addition of IGFI. B, cell counting assay for inhibition of proliferation of MCF cells in serum upon treatment with gml EM, IR, or H antibodies.C and D, inhibition of IGFI or serumstimulated proliferation and survival of breast cancer MCF by gml EM.DISCUSSION A monoclonal antibody EM has been developed that binds to the human IGFI receptor with a high affinity and neutralizes the function of IGFIR in cancer cells.EM inhibits the Thujone binding of IGFI to IGFIR in cells, blocks the activation of IGFIR and downstream signaling pathways, and suppresses the biological effects mediated by IGFIR.Inhibition of IGFIR function by EM resulted in significant anticancer activity both in vitro, where EM inhibited the growth of diverse cancer cell lines, and in vivo, where EM treatment suppressed the growth of a pancreatic cancer xenograft.In addition to blocking the binding of IGFI to IGFIR, EM treatment of cells at C causes significant downregulation of IGFIR levels, resulting in a decreasein hand a decrease in h.Similar downregulation of IGFIR at C was previously reported. ANTIINSULINLIKE GROWTH FACTOR I RECEPTOR ANTIBODY H, NCIH cells in serumfree medium lacking exogenously added IGFI or IGFII, as indicated by the lower MTT signals obtained upon EM treatment than for the untreated cells.The EM antibody, therefore, also provides a useful tool for testing the role of autocrine IGFIR signaling in the growth of cancer cell lines.Thus, the superior inhibition observed with EM is likely because of the high affinity of EM and strong inhibition of IGFI binding to IGFIR, rather than an enhanced ability to downregulate IGFIR levels.However, it is difficult to predict the biological consequences of inhibitory activity in shortterm signal transduction experiments because even a low level of IGFIR signaling could be above a minimal threshold level required for the proliferation and survival of cells.The effect of the antibody exposure in a longterm assay of proliferation and survival of cancer cells, therefore, may be a better measure of potential in vivo biological effects.In this regard, EM was significantly more inhibitory than the other three antibodies tested in a day assay of proliferation and survival of MCF breast cancer cells.EM inhibited the IGFI, IGFII, and serumstimulated proliferation and survival of the large majority of cancer cell lines tested, confirming the important role of IGFIR in cancer cell growth.

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