Inhibition of mTOR with Everolimus (RAD001) and Silencing by Vascular Endothelial Cell Growth Factor Specific siRNA Induces Additive Antitumor Activity in Multiple Myeloma Cells,

Angiogenesis plays an important role in the pathogenesis and progression in multiple myeloma (MM). MM cells secrete vascular endothelial growth factor (VEGF), which further promotes proliferation of the tumor cells. Several RNA interference (RNAi) methodologies are rapidly being established and hold promise to specifically inhibit gene expression in mammals. RNAi is the sequence-specific, posttranscription gene silencing methods initiated by double-stranded RNAs, which are homologous to the gene being suppressed. Mammalian target of rapamycin (mTOR) is an essential part of tumour growth being capable of integrating proliferative, antiapoptotic and angiogenic signalling by connecting VEGF, hypoxia-inducible factor 1 (HIF-1) and HER family receptors. Several reports have demonstrated the anti-tumor activity of everolimus (RAD001), an mTOR inhibitor, in a variety of cancers, including various leukemias and lymphomas.

Therefore, we evaluated the anti-myeloma effect of VEGF siRNA silencing in MM cells and whether it can be augmented by the additional application of everolimus.

Methods and Results: After transfection with VEGF siRNA we observed a reduction of VEGF expression in all studied cell lines: OPM-2, RPMI-8226, INA-6, Jurkat, Raji and Karpas-299, as well as in cells of MM- and lymphoma patients. Next, using the MM cell line OPM-2 we studied the time courses of VEGF siRNA transfection in order to investigate the knock-down efficiency of VEGF expression. The efficiency of VEGF siRNA transfection in treated OPM-2 cells showed a reduction of 75.5% VEGF protein levels after 24 h compared to the untreated OPM-2 cells (p<0.001). Further, VEGF siRNA both significantly induced apoptosis and inhibited proliferation in OPM-2 cells (p<0.001), RPMI-8226 (p<0.001), and in INA-6 (p<0.01) versus controls. To assess whether everolimus treatment of OPM-2 and RPMI-8226 affects the viability of these cells, cells were treated with various doses (1–20 nM) of everolimus for 24 hours, harvested, and analyzed for cell viability by MTT assay. Everolimus significantly decreased the viability of OPM-2 cells (IC₅₀ = 5.9 nM) and of RPMI-8226 cells (IC₅₀ = 0.6 nM), respectively. Everolimus and siRNA both together might to reduce the VEGF gene expression up to 33% (p<0.001) compare to siRNA VEGF (61%) alone or everolimus alone (39%), that demonstrated additive effects of everolimus and siRNA.

These findings suggest that mTOR inhibition and silencing by VEGF specific siRNA may be associated with an additive antitumor activity and might be a suitable target for new therapeutic strategies using RNA interference in MM.

No relevant conflicts of interest to declare.