with a variety of intracellular client proteins involved in cell growth

with a variety of intracellular client proteins involved in cell growth, differentiation and survival, to facilitate their folding, activity, intracellular localization and proteolytic turnover. Increased expression of Hsp90 is a common feature of hematological malignancies and its correct functioning is crucial for the FGFR Signaling Pathway survival of myelomatous plasma cells, because of the requirement of an appropriate chaperon machinery to manage the large amounts of proteins synthesized by these cells. These features make Hsp90 a potential target for anticancer drug development. Geldanamycin , a benzoquinone ansamycin antibiotic, interferes with the action of the Hsp90 leading to the degradation of Hsp90 client proteins.
Since many of these client proteins are oncogenic proteins, GA inhibits the proliferation of cancer cells and shows anticancer activity in experimental animals. However due to poor aqueous solubility and liver toxicity, GA has not moved forward in clinical trials. 17DMAG is a water soluble geldanamycin derivative that has potent inhibitory effects on cell proliferation Lenalidomide in cultured tumor cell lines and in vivo xenografts and is currently in phase I clinical trials. 17DMAG has been shown to induce apoptosis in MM cell lines and in primary MM cells, even in the presence of bone marrow stromal cells , which normally protect those cells from cell death . On the other hand, one of the key characteristics that distinguish myeloma plasma cells as a therapeutic target is the large quantity of monoclonal antibodies they synthesize and secrete.
Recently, it was reported that autophagy, an intracellular protein degradation system required for normal turnover of cellular components and for the starvation response, is activated upon endoplasmic reticulum stress as a defensive mechanism for survival . It functions fungus as an additional pathway that might protect MM cells against toxic misfolded proteins. In this study, we show for the first time that the Hsp90 inhibitor 17DMAG induces apoptosis and autophagy in human multiple myeloma cell lines. Interestingly, autophagy inhibition enhances 17DMAG induced apoptosis by amitochondria operated pathway. These data support the use of Hsp90 inhibitors and autophagy inhibition as a combination therapy in MM.Cells were treated in 6 well plates as indicated in the figure legends.
After treatment, cells were washed with phosphate buffered saline , fixed in cold 70% ethanol and then stained with propidium iodide while treating with RNAse. Quantitative analysis of sub G1 cells was carried out in a FACSCalibur cytometer using the Cell Quest software . Autophagic flux was determined by measuring the conversion of the microtubule associated protein light chain from the cytosolic LC3 I to the autophagosome bound LC3 II form in the presence and absence of lysosomal protease inhibitors . Autophagy was also determined by measuring p62/SQSTM1 and Beclin 1 protein levels. To induce starvation mediated autophagy cells were washed three times in phosphate buffer before they were amino acid starved for 5 h in Hanks’ balanced salt solution .To examine the effect of the Hsp90 inhibitor 17DMAG in the proliferation of human multiple myeloma cells, cultures of the MM.1S cell line were incubated in the presence of differents.