17-Allylamino-17-demethoxygeldanamycin induces downregulation of critical Hsp90 protein clients and results in cell cycle arrest and apoptosis of human urinary bladder cancer cells.
BACKGROUND
17-Allylamino-17-demethoxygeldanamycin (17-AAG), a benzoquinone ansamycin antibiotic, specifically targets heat shock protein 90 (Hsp90) and interferes with its function as a molecular chaperone that maintains the structural and functional integrity of various protein clients involved in cellular signaling. In this study, we have investigated the effect of 17-AAG on the regulation of Hsp90-dependent signaling pathways directly implicated in cell cycle progression, survival and motility of human urinary bladder cancer cell lines.


METHODS
We have used MTT-based assays, FACS analysis, Western blotting, semi-quantitative RT-PCR, immunocytochemistry and scratch-wound assay in RT4, RT112 and T24 human urinary bladder cancer cell lines.


RESULTS
We have demonstrated that, upon 17-AAG treatment, bladder cancer cells are arrested in the G1 phase of the cell cycle and eventually undergo apoptotic cell death in a dose-dependent manner. Furthermore, 17-AAG administration was shown to induce a pronounced downregulation of multiple Hsp90 protein clients and other downstream effectors, such as IGF-IR, Akt, IKK-α, IKK-β, FOXO1, ERK1/2 and c-Met, resulting in sequestration-mediated inactivation of NF-κB, reduced cell proliferation and decline of cell motility.


CONCLUSIONS
In total, we have clearly evinced a dose-dependent and cell type-specific effect of 17-AAG on cell cycle progression, survival and motility of human bladder cancer cells, due to downregulation of multiple Hsp90 clients and subsequent disruption of signaling integrity.
Karkoulis
Panagiotis K
PK
Laboratory of Environmental Mutagenesis and Carcinogenesis, Institute of Biology, National Center for Scientific Research (NCSR) Demokritos, 15310 Athens, Greece.
Stravopodis
Dimitrios J
DJ
Margaritis
Lukas H
LH
Voutsinas
Gerassimos E
GE
D017209:Apoptosis / Q000187:drug effects*; D016227:Benzoquinones / Q000494:pharmacology*; D015153:Blotting, Western; D002448:Cell Adhesion / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects*; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D014945:Wound Healing / Q000187:drug effects
D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D012333:RNA, Messenger; C112765:tanespimycin