Abstract
Considering the role of proto-oncogene c-Met (c-Met) in oncogenesis, we examined the effects of the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), and two NDRG1-inducing thiosemicarbazone-based agents, Dp44mT and DpC, on c-Met expression in DU145 and Huh7 cells. NDRG1 silencing without Dp44mT and DpC up-regulated c-Met expression, demonstrating that NDRG1 modulates c-Met levels. Dp44mT and DpC up-regulated NDRG1 by an iron-dependent mechanism and decreased c-Met levels, c-Met phosphorylation, and phosphorylation of its downstream effector, GRB2-associated binding protein 1 (GAB1). However, incubation with Dp44mT and DpC after NDRG1 silencing or silencing of the receptor tyrosine kinase inhibitor, mitogen-inducible gene 6 (MIG6), decreased c-Met and its phosphorylation, suggesting NDRG1- and MIG6-independent mechanism(s). Lysosomal inhibitors rescued the Dp44mT- and DpC-mediated c-Met down-regulation in DU145 cells. Confocal microscopy revealed that lysosomotropic agents and the thiosemicarbazones significantly increased co-localization between c-Met and lysosomal-associated membrane protein 2 (LAMP2). Moreover, generation of c-Met C-terminal fragment (CTF) and its intracellular domain (ICD) suggested metalloprotease-mediated cleavage. In fact, Dp44mT increased c-Met CTF while decreasing the ICD. Dp44mT and a γ-secretase inhibitor increased cellular c-Met CTF levels, suggesting that Dp44mT induces c-Met CTF levels by increasing metalloprotease activity. The broad metalloprotease inhibitors, EDTA and batimastat, partially prevented Dp44mT-mediated down-regulation of c-Met. In contrast, the ADAM inhibitor, TIMP metallopeptidase inhibitor 3 (TIMP-3), had no such effect, suggesting c-Met cleavage by another metalloprotease. Notably, Dp44mT did not induce extracellular c-Met shedding that could decrease c-Met levels. In summary, the thiosemicarbazones Dp44mT and DpC effectively inhibit oncogenic c-Met through lysosomal degradation and metalloprotease-mediated cleavage.
Highlights
Considering the role of proto-oncogene c-Met (c-Met) in oncogenesis, we examined the effects of the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), and two NDRG1-inducing thiosemicarbazone-based agents, Dp44mT and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), on c-Met expression in DU145 and Huh7 cells
Considering the role of proto-oncogene c-Met (c-Met) in oncogenesis, we examined the effects of the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), and two NDRG1-inducing thiosemicarbazone-based agents, Dp44mT and DpC, on c-Met expression in DU145 and Huh7 cells
We previously reported that the downregulation of EGFR was mediated by NDRG1 through its ability to directly associate with the EGFR inhibitor, mitogen-inducible gene-6 (MIG6), after genetic up-regulation of NDRG1 or after incubation of cells with thiosemicarbazones, which pharmacologically induce NDRG1 [26, 27]
Summary
The initial aim of this investigation was to examine the effect of NDRG1 silencing on c-Met expression in cancer cells. By examining c-Met levels in Huh cells, incubation with DFO, Dp44mT, or DpC induced a significant decrease in c-Met protein levels relative to the Control in the presence or absence of HGF (Fig. 2, C and Di). Co-incubation of Dp44mT (5 M) and batimastat (5 M), largely prevented the down-regulation of c-Met, leading to significantly higher c-Met levels compared with Dp44mT alone (Fig. 8, A and B) These results suggest the ability of batimastat to inhibit metalloprotease activity prevents the loss of c-Met. Studies examined the effect of TIMP-3, which has been demonstrated to be an inhibitor of metalloprotease activity that potently inhibits c-Met shedding by the antibody, DN30, via blocking ADAM10 proteolytic activity [16]. Dation that were differentially observed depending on the cell type examined
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