monensin and Multiple-Myeloma

The Golgi is a dynamic organelle whose correct assembly is crucial for cellular homeostasis. Perturbations in Golgi structure are associated with numerous disorders from neurodegeneration to cancer. However, whether and how dispersal of the Golgi apparatus is actively regulated under stress, and the consequences of Golgi dispersal, remain unknown. Here we demonstrate that 26S proteasomes are associated with the cytosolic surface of Golgi membranes to facilitate Golgi Apparatus-Related Degradation (GARD) and degradation of GM130 in response to Golgi stress. The degradation of GM130 is dependent on p97/VCP and 26S proteasomes, and required for Golgi dispersal. Finally, we show that perturbation of Golgi homeostasis induces cell death of multiple myeloma in vitro and in vivo, offering a therapeutic strategy for this malignancy. Taken together, this work reveals a mechanism of Golgi-localized proteasomal degradation, providing a functional link between proteostasis control and Golgi architecture, which may be critical in various secretion-related pathologies.

Topics: Animals; Apoptosis; Autoantigens; Cell Line, Tumor; Disease Models, Animal; Golgi Apparatus; HEK293 Cells; Humans; Intracellular Membranes; Ionophores; Membrane Proteins; Mice; Monensin; Multiple Myeloma; Proteasome Endopeptidase Complex; Proteolysis; Proteostasis; Ubiquitination; Valosin Containing Protein

Previously, we showed that monensin, Na+ ionophore, potently inhibited the growth of acute myelogenous leukemia and lymphoma cells. Here, we investigated the antiproliferative effect of monensin on human myeloma cell lines. Monensin significantly inhibited the proliferation of myeloma cell lines examined with IC50 of about 1 micro M. Cell cycle analysis indicated that monensin induced a G1 and/or a G2-M phase arrest in these cell lines. To address the mechanism of the antiproliferative effect of monensin, we examined the effect of this drug on cell cycle-related proteins in NCI-H929 cells. Monensin decreased the levels of CDK2, CDK6, cdc2, cyclin A, cyclin B1, cyclin D1 and cyclin E proteins but did not alter CDK4 protein. While p21 was increased by monensin, p27 was not. In addition, monensin markedly enhanced the binding of p21 with CDK6 and cdc2. Furthermore, the activities of CDK2- and CDK6-associated kinases were reduced in association with hypophosphorylation of Rb protein. The activity of cdc2-associated kinase was decreased, which was accompanied by reduction of cdc25C phosphatase. Also, monensin induced apoptosis in myeloma cells, as evidenced by annexin V binding assay and flow cytometric detection of sub-G1 DNA content. This apoptotic process was associated with down-regulation of Bcl-2, loss of mitochondria transmembrane potential (Deltapsim) and an increase of caspase-3 activity. In addition, monensin caused the up-regulation of ERK and p38 kinase activities. Taken together, these results have demonstrated for the first time that monensin potently inhibited the proliferation of human myeloma cell lines, especially NCI-H929 cells, via cell cycle arrest in association with p21 and apoptosis.

Topics: Antifungal Agents; Apoptosis; Blotting, Western; CDC2 Protein Kinase; cdc25 Phosphatases; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Cell Separation; Cyclin D1; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Dose-Response Relationship, Drug; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Flow Cytometry; G2 Phase; Humans; Inhibitory Concentration 50; Membrane Potentials; Microfilament Proteins; Mitochondria; Mitosis; Models, Chemical; Monensin; Multiple Myeloma; Muscle Proteins; Precipitin Tests; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Time Factors

The murine plasma cell line MOPC 315 efficiently targets newly synthesized acid hydrolases to lysosomes in spite of a marked deficiency in the level of the mannose 6-phosphate receptor (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). To better understand the routing of lysosomal enzymes in this cell line, pulse-chase experiments were performed with [2-3H]mannose and [35S]methionine followed by immunoprecipitation of beta-glucuronidase and IgA. By 3 h of chase, essentially all of the newly synthesized beta-glucuronidase had undergone proteolytic processing, suggesting that the molecules had reached lysosomes. At this time 30% of the pulse-labeled IgA was still intracellular. The oligosaccharides on the intracellular IgA were of the high mannose-type, while the secreted IgA contained processed, complex-type oligosaccharides. This indicates that the intracellular IgA was still in the endoplasmic reticulum or an early region of the Golgi complex when all of the beta-glucuronidase had reached lysosomes. Therefore, beta-glucuronidase and IgA must exit from the endoplasmic reticulum or the early Golgi complex at different rates, a finding that is inconsistent with bulk phase movement of these proteins from the endoplasmic reticulum to the trans Golgi complex. The addition of the ionophore monensin greatly slows the rate of IgA secretion from MOPC 315 cells and the molecules secreted have incompletely processed oligosaccharides. In contrast, monensin only slightly delays the transport of newly synthesized beta-glucuronidase to lysosomes and causes no significant alteration in the extent of oligosaccharide phosphorylation, a process that appears to occur in the early (cis) Golgi complex. However, the labeled beta-glucuronidase was deficient in sialylated, phosphorylated hybrid oligosaccharides whose biosynthesis requires the action of late stage oligosaccharide processing enzymes assumed to be localized in the trans Golgi complex.

Topics: Animals; Cell Line; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Glucuronidase; Golgi Apparatus; Immunoglobulin A; Lysosomes; Mice; Monensin; Multiple Myeloma; Receptor, IGF Type 2; Receptors, Cell Surface