Abstract
Background17-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.MethodsWe 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.ResultsWe 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.ConclusionsIn 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.
Highlights
Urinary bladder cancer is the fifth most common malignancy in the industrialized world and the second most frequent malignancy of the genitourinary tract, demonstrating high heterogeneity and differential response to clinical treatment [1,2]
Here, we have thoroughly studied the effects of heat shock protein 90 (Hsp90) inhibition by 17-AAG on the Hsp90assisted signaling repertoire associated with cell cycle progression, apoptosis and motility in three human urinary bladder cancer cell lines of different malignancy grade, namely RT4, RT112 and T24
Polyclonal and monoclonal antibodies against Caspase-8, Caspase-9, Caspase-3, PARP, Lamin A/C, phospho-Akt (Ser473), phospho-Akt (Thr308), Akt, phospho-IGF-ΙRb (Tyr1131), IGF-ΙRa, FOXO, phospho-FOXO, phosphoIKKa/b (Ser180/Ser181), IKKa, IKKb, phospho-p44/42 (Thr202/Tyr204), p44/42, a-tubulin, phospho-c-Met (Tyr1234/Tyr1235), c-Met, Carboxyl terminus of Hsp70 interacting protein (CHIP) and pan-actin were purchased from Cell Signaling Technology Inc. (Hertfordshire, UK), whereas antibodies against Hsp90a/b, Hsp70, Cdk4, pRb, E2F1 and NF-B (p65) were supplied by Santa Cruz Biotechnology Inc. (California, USA)
Summary
Urinary bladder cancer is the fifth most common malignancy in the industrialized world and the second most frequent malignancy of the genitourinary tract, demonstrating high heterogeneity and differential response to clinical treatment [1,2]. Over-expression and/or presence of mutations in a variety of Hsp protein clients during cancer initiation is associated with a requirement for increased Hsp levels in order to maintain the active conformations and functional integrities of these oncogenic molecules. In this frame, Hsp is a key molecule in the conformational maturation of several bona fide oncogenic signaling proteins, such as HER2/ErbB2, Akt, Met, Raf, p53 and HIF-1a [10,12]. Due to the dependence of cancer cells upon specific Hsp oncogenic protein clients, inhibition of Hsp was shown to be able to negatively interfere with a number of important signaling pathways involved in cell development, proliferation, survival and motility, arousing significant interest in the field of cancer therapeutics [13]
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