Tumor Biology

, Volume 37, Issue 5, pp 6861–6873 | Cite as

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

  • Panagiotis K. Karkoulis
  • Dimitrios J. Stravopodis
  • Gerassimos E. Voutsinas
Original Article
  • 155 Downloads

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.

Keywords

17-DMAG Apoptosis Bladder cancer Hsp90 Metastasis Signaling 

Notes

Acknowledgments

We would like to thank Dr. Dimitrios Kletsas and Dr. Harris Pratsinis (Laboratory of Cell Proliferation and Ageing, Institute of Biosciences and Applications, NCSR “Demokritos”, Athens, Greece) for their assistance in cell cycle analysis. We are also grateful to Dr. Eumorphia G. Konstantakou (Post-doctoral Research Fellow, Department of Cell Biology and Biophysics, Faculty of Biology, National, and Kapodistrian University of Athens, Athens, Greece) for her valuable help in cell culture maintenance and sqPCR experiments. Finally, we must thank Dr. Athanassios D. Velentzas (Post-doctoral Research Fellow, Department of Cell Biology and Biophysics, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece) for his valuable help and technical support in confocal laser scanning microscopy and imaging. This work was performed in the framework of “Target Identification for Disease Diagnosis and Treatment (DIAS)” project within GSRT’s KRIPIS action, funded by Greece and the European Regional Development Fund of the European Union under the O.P. Competitiveness and Entrepreneurship, NSRF 2007–2013. Financial support to DJS and GEV was provided by the Empeirikeion Foundation (30-12-2009/Athens, Greece). Implementation of this work was also supported by additional funding kindly provided by The American College of Greece.

Compliance with Ethical Standards

Conflicts of Interest

None

Supplementary material

13277_2015_4544_MOESM1_ESM.pdf (2.9 mb)
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_MOESM2_ESM.pdf (3.1 mb)
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)

References

  1. 1.
    Stravopodis DJ, Margaritis LH, Voutsinas GE. Drug-mediated targeted disruption of multiple protein activities through functional inhibition of the Hsp90 chaperone complex. Curr Med Chem. 2007;14:3122–38.CrossRefPubMedGoogle Scholar
  2. 2.
    Picard D. Heat-shock protein 90, a chaperone for folding and regulation. Cell Mol Life Sci. 2002;59:1640–8.CrossRefPubMedGoogle Scholar
  3. 3.
    Sherman M, Multhoff G. Heat shock proteins in cancer. Ann N Y Acad Sci. 2007;1113:192–201.CrossRefPubMedGoogle Scholar
  4. 4.
    Whitesell L, Lindquist SL. HSP90 and the chaperoning of cancer. Nat Rev Cancer. 2005;5:761–72.CrossRefPubMedGoogle Scholar
  5. 5.
    Bagatell R, Whitesell L. Altered Hsp90 function in cancer: a unique therapeutic opportunity. Mol Cancer Ther. 2004;3:1021–30.CrossRefPubMedGoogle Scholar
  6. 6.
    Taldone T, Zatorska D, Patel PD, Zong H, Rodina A, Ahn JH, et al. Design, synthesis, and evaluation of small molecule Hsp90 probes. Bioorg Med Chem. 2011;19:2603–14.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Jego G, Hazoume A, Seigneuric R, Garrido C. Targeting heat shock proteins in cancer. Cancer Lett. 2013;332:275–85.CrossRefPubMedGoogle Scholar
  8. 8.
    Miyata Y, Nakamoto H, Neckers L. The therapeutic target Hsp90 and cancer hallmarks. Curr Pharm Des. 2013;19:347–65.CrossRefPubMedGoogle Scholar
  9. 9.
    Workman P, Burrows F, Neckers L, Rosen N. Drugging the cancer chaperone HSP90: combinatorial therapeutic exploitation of oncogene addiction and tumor stress. Ann N Y Acad Sci. 2007;1113:202–16.CrossRefPubMedGoogle Scholar
  10. 10.
    Siegel R, Naishadham D, Jemal A. Cancer statistics. CA Cancer J Clin. 2013;63:11–30.CrossRefPubMedGoogle Scholar
  11. 11.
    Mitra AP, Datar RH, Cote RJ. Molecular staging of bladder cancer. BJU Int. 2005;96:7–12.CrossRefPubMedGoogle Scholar
  12. 12.
    Wu XR. Urothelial tumorigenesis: a tale of divergent pathways. Nat Rev Cancer. 2005;5:713–25.CrossRefPubMedGoogle Scholar
  13. 13.
    Goebell PJ, Knowles MA. Bladder cancer or bladder cancers? genetically distinct malignant conditions of the urothelium. Urol Oncol. 2010;28:409–28.CrossRefPubMedGoogle Scholar
  14. 14.
    Bellmunt J, Petrylak DP. New therapeutic challenges in advanced bladder cancer. Semin Oncol. 2012;39:598–607.CrossRefPubMedGoogle Scholar
  15. 15.
    Garcia JA, Dreicer R. Systemic chemotherapy for advanced bladder cancer: update and controversies. J Clin Oncol. 2006;24:5545–51.CrossRefPubMedGoogle Scholar
  16. 16.
    Sonpavde G, Sternberg CN, Rosenberg JE, Hahn NM, Galsky MD, Vogelzang NJ. Second-line systemic therapy and emerging drugs for metastatic transitional-cell carcinoma of the urothelium. Lancet Oncol. 2011;11:861–70.CrossRefGoogle Scholar
  17. 17.
    Comprehensive molecular characterization of urothelial bladder carcinoma. Nature 2014; 507:315-322.Google Scholar
  18. 18.
    Kamal A, Thao L, Sensintaffar J, Zhang L, Boehm MF, Fritz LC, et al. A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors. Nature. 2003;425:407–10.CrossRefPubMedGoogle Scholar
  19. 19.
    Okamoto J, Mikami I, Tominaga Y, Kuchenbecker KM, Lin YC, Bravo DT, et al. Inhibition of Hsp90 leads to cell cycle arrest and apoptosis in human malignant pleural mesothelioma. J Thorac Oncol. 2008;3:1089–95.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Basso AD, Solit DB, Munster PN, Rosen N. Ansamycin antibiotics inhibit Akt activation and cyclin D expression in breast cancer cells that overexpress HER2. Oncogene. 2002;21:1159–66.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Garcia-Morales P, Carrasco-Garcia E, Ruiz-Rico P, Martinez-Mira R, Menendez-Gutierrez MP, Ferragut JA, et al. Inhibition of Hsp90 function by ansamycins causes downregulation of cdc2 and cdc25c and G (2)/M arrest in glioblastoma cell lines. Oncogene. 2007;26:7185–93.CrossRefPubMedGoogle Scholar
  22. 22.
    Hostein I, Robertson D, DiStefano F, Workman P, Clarke PA. Inhibition of signal transduction by the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in cytostasis and apoptosis. Cancer Res. 2001;61:4003–9.PubMedGoogle Scholar
  23. 23.
    Cardillo MR, Sale P, Di Silverio F. Heat shock protein-90, IL-6 and IL-10 in bladder cancer. Anticancer Res. 2000;20:4579–83.PubMedGoogle Scholar
  24. 24.
    Yoshida S, Koga F, Tatokoro M, Kawakami S, Fujii Y, Kumagai J, et al. Low-dose Hsp90 inhibitors tumor-selectively sensitize bladder cancer cells to chemoradiotherapy. Cell Cycle. 2011;10:4291–9.CrossRefPubMedGoogle Scholar
  25. 25.
    McCollum AK, Teneyck CJ, Sauer BM, Toft DO, Erlichman C. Up-regulation of heat shock protein 27 induces resistance to 17-allylamino-demethoxygeldanamycin through a glutathione-mediated mechanism. Cancer Res. 2006;66:10967–75.CrossRefPubMedGoogle Scholar
  26. 26.
    Clarke PA, Hostein I, Banerji U, Stefano FD, Maloney A, Walton M, et al. Gene expression profiling of human colon cancer cells following inhibition of signal transduction by 17-allylamino-17-demethoxygeldanamycin, an inhibitor of the hsp90 molecular chaperone. Oncogene. 2000;19:4125–33.CrossRefPubMedGoogle Scholar
  27. 27.
    Stravopodis DJ, Karkoulis PK, Konstantakou EG, Melachroinou S, Lampidonis AD, Anastasiou D, et al. Grade-dependent effects on cell cycle progression and apoptosis in response to doxorubicin in human bladder cancer cell lines. Int J Oncol. 2009;34:137–60.PubMedGoogle Scholar
  28. 28.
    Konstantakou EG, Voutsinas GE, Karkoulis PK, Aravantinos G, Margaritis LH, Stravopodis DJ. Human bladder cancer cells undergo cisplatin-induced apoptosis that is associated with p53-dependent and p53-independent responses. Int J Oncol. 2009;35:401–16.PubMedGoogle Scholar
  29. 29.
    Beck R, Verrax J, Gonze T, Zappone M, Pedrosa RC, Taper H, et al. Hsp90 cleavage by an oxidative stress leads to its client proteins degradation and cancer cell death. Biochem Pharmacol. 2009;77:375–83.CrossRefPubMedGoogle Scholar
  30. 30.
    Shen SC, Yang LY, Lin HY, Wu CY, Su TH, Chen YC. Reactive oxygen species-dependent HSP90 protein cleavage participates in arsenical As (+3) - and MMA (+3)-induced apoptosis through inhibition of telomerase activity via JNK activation. Toxicol Appl Pharmacol. 2008;229:239–51.CrossRefPubMedGoogle Scholar
  31. 31.
    Lang SA, Moser C, Gaumann A, Klein D, Glockzin G, Popp FC, et al. Targeting heat shock protein 90 in pancreatic cancer impairs insulin-like growth factor-I receptor signaling, disrupts an interleukin-6/signal-transducer and activator of transcription 3/hypoxia-inducible factor-1alpha autocrine loop, and reduces orthotopic tumor growth. Clin Cancer Res. 2007;13:6459–68.CrossRefPubMedGoogle Scholar
  32. 32.
    Karkoulis PK, Stravopodis DJ, Margaritis LH, Voutsinas GE. 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. BMC Cancer. 2010;10:481.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Karkoulis PK, Stravopodis DJ, Konstantakou EG, Voutsinas GE. Targeted inhibition of heat shock protein 90 disrupts multiple oncogenic signaling pathways, thus inducing cell cycle arrest and programmed cell death in human urinary bladder cancer cell lines. Cancer Cell Int. 2013;13:11.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Xie Q, Gao CF, Shinomiya N, Sausville E, Hay R, Gustafson M, et al. Geldanamycins exquisitely inhibit HGF/SF-mediated tumor cell invasion. Oncogene. 2005;24:3697–707.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Panagiotis K. Karkoulis
    • 1
  • Dimitrios J. Stravopodis
    • 2
  • Gerassimos E. Voutsinas
    • 1
  1. 1.Laboratory of Environmental Mutagenesis and CarcinogenesisInstitute of Biosciences and Applications, National Center for Scientific Research (NCSR) “Demokritos”AthensGreece
  2. 2.Department of Cell Biology and Biophysics, Faculty of BiologyNational and Kapodistrian University of AthensAthensGreece

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