betadex and 5-dimethylamiloride

Hsp60, a Group I mitochondrial chaperonin, is classically considered an intracellular chaperone with residence in the mitochondria; nonetheless, in the last few years it has been found extracellularly as well as in the cell membrane. Important questions remain pertaining to extracellular Hsp60 such as how generalized is its occurrence outside cells, what are its extracellular functions and the translocation mechanisms that transport the chaperone outside of the cell. These questions are particularly relevant for cancer biology since it is believed that extracellular chaperones, like Hsp70, may play an active role in tumor growth and dissemination.. Since cancer cells may undergo necrosis and apoptosis, it could be possible that extracellular Hsps are chiefly the result of cell destruction but not the product of an active, physiological process. In this work, we studied three tumor cells lines and found that they all release Hsp60 into the culture media by an active mechanism independently of cell death. Biochemical analyses of one of the cell lines revealed that Hsp60 secretion was significantly reduced, by inhibitors of exosomes and lipid rafts.. Our data suggest that Hsp60 release is the result of an active secretion mechanism and, since extracellular release of the chaperone was demonstrated in all tumor cell lines investigated, our observations most likely reflect a general physiological phenomenon, occurring in many tumors.

Topics: Acetylcholinesterase; Amiloride; Apoptosis; beta-Cyclodextrins; Blotting, Western; Cell Line; Cell Line, Tumor; Cell Survival; Chaperonin 60; Culture Media, Conditioned; Exosomes; Extracellular Space; Humans; K562 Cells; Microscopy, Electron, Transmission; Neoplasms

The heat shock proteins (HSP) are a highly conserved family of proteins with critical functions in protein folding, protein trafficking, and cell signaling. These proteins also protect the cell against injury. HSP60 has been found in the extracellular space and has been identified in the plasma of some individuals. HSP60 is thought to be a "danger signal" to the immune system and is also highly immunogenic. Thus extracellular HSP60 is possibly toxic to the cell. The mechanism by which HSP60 is released into the extracellular space is unknown, as is whether it is released by cardiac myocytes. We investigated several different pathways controlling protein release including the classic, Golgi-mediated pathway. We found that HSP60 is released via exosomes, and that within the exosome, HSP60 is tightly attached to the exosome membrane.

Topics: Amiloride; Animals; beta-Cyclodextrins; Brefeldin A; Caveolin 1; Cell Hypoxia; Chaperonin 60; Golgi Apparatus; HSC70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Male; Membrane Microdomains; Myocytes, Cardiac; Protein Transport; Rats; Rats, Sprague-Dawley; Secretory Vesicles; Subcellular Fractions