Targeting MEK/MAPK Signal Transduction Module Potentiates Arsenic Trioxide (ATO)-Induced Apoptosis in Multiple Myeloma Cells through Multiple Signaling Pathways.

Multiple Myeloma (MM) cells are extremely resistant to apoptosis and currently new potential drug combinations are under investigation. We have shown that the combined treatment with the MEK1/2 inhibitor PD184352 (PD) and Arsenic Trioxide (ATO) resulted in the synergistic (Combination Index <1.0) induction of apoptosis in 7 human myeloma cell lines (HMCLs: XG1, XG6, OPM2, JJN3, RPMI, H929, Sultan) analyzed, irrespective of their p53 status. The combined treatment was also a highly potent inducer of apoptosis and mitochondrial damage in the majority of the primary multiple myeloma (MM) cell samples ex vivo analyzed at different disease stage (9 out of 12). Growth factors, IL-6 or insulin-like growth factor 1 (IGF-1), or a co-culture system with bone marrow stromal cells (BMSCs) failed to confer resistance to this combination regimen. The combination PD/ATO had a minimal effect on normal B cells in vitro. By investigating the molecular mechanisms involved in MM cells PD/ATO-induced apoptosis, we found that co-treatment with PD strikingly elevated the (DR4+DR5)/(DcR1+DcR2) tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors ratio, caspase-8 activation, Bid fragmentation, mitochondrial depolarization and caspase-9 activation of ATO-treated HMCLs that do not have a functional p53 pathway. In HMCLs carrying a functional p53 pathway, the treatment with PD greatly enhanced the ATO-induced p53 accumulation (two fold increase) and p73, a p53 paralogue, cooperated with p53 in the pro-apoptotic p53/p73 target genes up regulation, caspase-9, -3 activation and apoptosis induction; in these HMCLs the selective down-regulation of p53 or p73 demonstrated that both have a biological relevance in PD/ATO-induced caspase-3 activation, PARP fragmentation and apoptosis. In HMCLs carrying a functional p53 the extrinsic caspase-8 mediated pathway was partially activated by PD/ATO treatment. We also demonstrated that, in MM cells carrying or not a functional p53 pathway, the combined treatment PD/ATO increased the level of the pro-apoptotic Bim (PD-mediated) and decreased its neutralizing anti-apoptotic protein Mcl-1 (ATO-mediated). The selective down-regulation of Bim significantly diminished caspase-8/-9/-3 cleavage/activation, PARP fragmentation and apoptosis of PD/ATO-treated MM cells, thereby indicating that Bim can play an important role not only in the intrinsic mitochondrial programmed cell death but also in the extrinsic caspase-8 mediated pathway. Accordingly, a physical interaction between Bim and DR4/DR5 TRAIL receptors in PD/ATO-treated MM cells carrying a non functional p53 was found by coimmunoprecipitation and Western blot studies. Our experiments have enlightened some relevant mechanisms that explain the apoptotic response of myeloma cells to ATO plus MEK inhibitor combination.