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11.12.2015 | Original Article | Ausgabe 5/2016

Tumor Biology 5/2016

17-DMAG induces heat shock protein 90 functional impairment in human bladder cancer cells: knocking down the hallmark traits of malignancy

Zeitschrift:
Tumor Biology > Ausgabe 5/2016
Autoren:
Panagiotis K. Karkoulis, Dimitrios J. Stravopodis, Gerassimos E. Voutsinas
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1007/​s13277-015-4544-2) contains supplementary material, which is available to authorized users.

Abstract

Heat shock protein 90 (Hsp90) is a molecular chaperone that maintains the structural and functional integrity of various protein clients involved in multiple oncogenic signaling pathways. Hsp90 holds a prominent role in tumorigenesis, as numerous members of its broad clientele are involved in the generation of the hallmark traits of cancer. 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) specifically targets Hsp90 and interferes with its function as a molecular chaperone, impairing its intrinsic ATPase activity and undermining proper folding of multiple protein clients. In this study, we have examined the effects of 17-DMAG on the regulation of Hsp90-dependent tumorigenic signaling pathways directly implicated in cell cycle progression, survival, and motility of human urinary bladder cancer cell lines. We have used MTT-based assays, FACS analysis, Western blotting, semiquantitative PCR (sqPCR), immunofluorescence, and scratch-wound assays in RT4 (p53wt), RT112 (p53wt), T24 (p53mt), and TCCSUP (p53mt) human urinary bladder cancer cell lines. We have demonstrated that, upon exposure to 17-DMAG, bladder cancer cells display prominent cell cycle arrest and commitment to apoptotic and autophagic cell death, in a dose-dependent manner. Furthermore, 17-DMAG administration induced pronounced downregulation of multiple Hsp90 protein clients and other downstream oncogenic effectors, therefore causing inhibition of cell proliferation and decline of cell motility due to the molecular “freezing” of critical cytoskeletal components. In toto, we have clearly demonstrated the dose-dependent and cell type-specific effects of 17-DMAG on the hallmark traits of cancer, appointing Hsp90 as a key molecular component in bladder cancer targeted therapy.

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Zusatzmaterial
Online Resource 1 Graphs of protein and mRNA transcript expression profiles, upon 24 h of 17-DMAG administration. A) Cdk4 densitometric quantification bars, denoting the drug-induced alterations of protein expression levels compared to control conditions, using total actin as protein of reference. B) Cyclin D1 densitometric mRNA expression profiles, in all four bladder cancer cell lines examined herein, in response to 24 h of 17-DMAG treatment, compared to control conditions, using GAPDH as gene of reference. Standard deviation values are depicted as error bars on top of each value. (PDF 2940 kb)
13277_2015_4544_MOESM1_ESM.pdf
Online Resource 2 Graphs of mRNA transcript expression profiles upon 24 h of 17-DMAG administration. cIAP1 (A) and Survivin (B) densitometric mRNA expression profiles, in all four bladder cancer cell lines examined herein, in response to 17-DMAG treatment, compared to control conditions, using GAPDH as gene of reference. Standard deviation values are depicted as error bars on top of each value. (PDF 3206 kb)
13277_2015_4544_MOESM2_ESM.pdf
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