benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and geldanamycin

Selective modulation of cell death is important for rational chemotherapy. By depleting Hsp90-client oncoproteins, geldanamycin (GA) and 17-allylamino-17-demethoxy-GA (17-AAG) (heat-shock protein-90-active drugs) render certain oncoprotein-addictive cancer cells sensitive to chemotherapy. Here we investigated effects of GA and 17-AAG in apoptosis-prone cells such as HL60 and U937. In these cells, doxorubicin (DOX) caused rapid apoptosis, whereas GA-induced heat-shock protein-70 (Hsp70) (a potent inhibitor of apoptosis) and G1 arrest without significant apoptosis. GA blocked caspase activation and apoptosis and delayed cell death caused by DOX. Inhibitors of translation and transcription and siRNA Hsp70 abrogated cytoprotective effects of GA. Also GA failed to protect HL60 cells from cytotoxicity of actinomycin D and flavopiridol (FL), inhibitors of transcription. We next compared cytoprotection by GA-induced Hsp70, caspase inhibitors (Z-VAD-fmk) and cell-cycle arrest. Whereas cell-cycle arrest protected HL60 cells from paclitaxel (PTX) but not from FL and DOX, Z-VAD-fmk prevented FL-induced apoptosis but was less effective against DOX and PTX. Thus, by inducing Hsp70, GA protected apoptosis-prone cells in unique and cell-type selective manner. Since GA does not protect apoptosis-reluctant cancer cells, we envision a therapeutic strategy to decrease side effects of chemotherapy without affecting its therapeutic efficacy.

Topics: Amino Acid Chloromethyl Ketones; Antibiotics, Antineoplastic; Apoptosis; Benzoquinones; Caspase 9; Caspase Inhibitors; Cell Cycle; Cell Line, Tumor; Cytoprotection; Dactinomycin; Doxorubicin; Enzyme Activation; Flavonoids; HSP70 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Paclitaxel; Piperidines; Protein Biosynthesis; RNA, Small Interfering; Transcriptional Activation

The geldanamycin-induced degradation of ErbB-2 produces a 23-kDa carboxyl-terminal fragment, which has been isolated and subjected to amino-terminal microsequencing. The obtained sequence indicates that the amino terminus of this fragment corresponds to Gly-1126 of ErbB-2. Analysis of the residues immediately before Gly-1126 suggests that cleavage may involve caspase activity. Site-directed mutagenesis of Asp-1125 in ErbB-2 prevents geldanamycin-provoked formation of the 23-kDa fragment, consistent with the requirement of this residue for caspase-dependent cleavage in known substrates. Also, the addition of the pan-caspase inhibitor Z-VAD-FMK blocks formation of the 23-kDa ErbB-2 fragment in cells exposed to geldanamycin. Interestingly, staurosporin and curcumin are also shown to provoke the degradation of ErbB-2 with formation of the 23-kDa carboxyl-terminal fragment. The generation of this fragment by staurosporin or curcumin is likewise blocked by caspase inhibition. Caspase inhibition does not prevent accelerated degradation of the 185-kDa native ErbB-2 in geldanamycin-treated cells but does significantly prevent staurosporin-stimulated metabolic loss of ErbB-2.

Topics: Amino Acid Chloromethyl Ketones; Animals; Benzoquinones; Caspase Inhibitors; Caspases; COS Cells; Curcumin; Cytoplasm; Enzyme Inhibitors; Glycine; Humans; Immunoblotting; Lactams, Macrocyclic; Mutagenesis, Site-Directed; Mutation; Protein Binding; Protein Structure, Tertiary; Quinones; Receptor, ErbB-2; Sequence Analysis, Protein; Staurosporine; Transfection; Tumor Cells, Cultured