Cancer Chemotherapy and Pharmacology

, Volume 56, Issue 2, pp 126–137 | Cite as

Comparison of 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17DMAG) and 17-allylamino-17-demethoxygeldanamycin (17AAG) in vitro: effects on Hsp90 and client proteins in melanoma models

  • Victoria Smith
  • Edward A. Sausville
  • Richard F. Camalier
  • Heinz-Herbert Fiebig
  • Angelika M. Burger
Original Article

Abstract

The heat shock protein Hsp90 is a potential target for drug discovery of novel anticancer agents. By affecting this protein, several cell signaling pathways may be simultaneously modulated. The geldanamycin analog 17AAG has been shown to inhibit Hsp90 and associated proteins. Its clinical use, however, is hampered by poor solubility and thus, difficulties in formulation. Therefore, a water-soluble derivative was desirable and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17DMAG) is such a derivative. Studies were carried out in order to evaluate the activity and molecular mechanism(s) of 17DMAG in comparison with those of 17-allylamino-demethoxygeldanamycin (17AAG). 17DMAG was found to be more potent than 17AAG in a panel of 64 different patient-derived tumor explants studied in vitro in the clonogenic assay. The tumor types that responded best included mammary cancers (six of eight), head and neck cancers (two of two), sarcomas (four of four), pancreas carcinoma (two of three), colon tumors (four of eight for 17AAG and six of eight for 17DMAG), and melanoma (two of seven). Bioinformatic comparisons suggested that, while 17AAG and 17DMAG are likely to share the same mode(s) of action, there was very little similarity with standard anticancer agents. Using three permanent human melanoma cell lines with differing sensitivities to 17AAG and 17DMAG (MEXF 276L, MEXF 462NL and MEXF 514L), we found that Hsp90 protein was reduced following treatment at a concentration associated with total growth inhibition. The latter occurred in MEXF 276L cells only, which are most sensitive to both compounds. The depletion of Hsp90 was more pronounced in cells exposed to 17DMAG than in those treated with 17AAG. The reduction in Hsp90 was associated with the expression of erbB2 and erbB3 in MEXF 276L, while erbB2 and erbB3 were absent in the more resistant MEXF 462NL and MEXF 514L cells. Levels of known Hsp90 client proteins such as phosphorylated AKT followed by AKT, cyclin D1 preceding cdk4, and craf-1 declined as a result of drug treatment in all three melanoma cell lines. However, the duration of drug exposure needed to achieve these effects was variable. All cell lines showed increased expression of Hsp70 and activated cleavage of PARP. No change in PI3K expression was observed and all melanoma cells were found to harbor the activating V599E BRAF kinase mutation. The results of our in vitro studies are consistent with both 17AAG and 17DMAG acting via the same molecular mechanism, i.e. by modulating Hsp90 function. Since 17DMAG can be formulated in physiological aqueous solutions, the data reported here strongly support the development of 17DMAG as a more pharmaceutically practicable congener of 17AAG.

Keywords

17DMAG 17AAG Hsp90 modulation Melanoma 

Abbreviations

17AAG

17-Allylamino-17-demethoxygeldanamycin

17DMAG

17-Dimethylaminoethylamino-17-demethoxygeldanamycin

5-FU

5-Fluorouracil

PBS

Phosphate-buffered saline

NCI

National Cancer Institute

DMSO

Dimethylsulfoxide

Hsp

Heat shock protein

MEXF

Melanoma xenograft established by Fiebig et al.

PARP

Poly-adenosine ribose polymerase

TGI

Total growth inhibition (no change vs initial cell number)

GI50

Growth-inhibitory concentration 50% compared to control

TCA

Tumor clonogenic assay

EDTA

Ethylenediaminetetraacetic acid

References

  1. 1.
    Maloney A, Workman P (2002) HSP90 as a new therapeutic target for cancer therapy: the story unfolds. Expert Opin Biol Ther 2:3Google Scholar
  2. 2.
    Hostein I, Robertson D, DiStefano F, Workman P, Clarke PA (2001) Inhibition of signal transduction by the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in cytostasis and apoptosis. Cancer Res 61:4003Google Scholar
  3. 3.
    Basso A, Solit D, Chiosis G, Giri B, Tsichlis P, Rosen N (2002) Akt forms an intracellular complex with heat shock protein 90 (Hsp90) and cdc37 and is destabilised by inhibitors of Hsp90 function. J Biol Chem 277:39858Google Scholar
  4. 4.
    Fujita N, Sato S, Ishida A, Tsuruo T (2002) Involvement of Hsp90 in signaling and stability of 3-phosphoinositide-dependent kinase. J Biol Chem 277:10346Google Scholar
  5. 5.
    Stebbins C, Russo A, Schnieder C, Rosen N, Hartl F, Pavletich N (1997) Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent. Cell 89:239Google Scholar
  6. 6.
    Prodromou C, Roe S, O’Brien R, Ladbury J, Piper P, Pearl L (1997) Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone. Cell 90:65Google Scholar
  7. 7.
    Page J, Heath J, Fulton R, Yalkowsky E, Tabibi E, Tomaszewski J, Smith A, Rodman L (1997) Comparison of geldanamycin (NSC-122750) and 17-allylaminogeldanamycin (NSC 330507D) toxicity in rats. Proc Annu Meet Am Assoc Cancer Res 38:308Google Scholar
  8. 8.
    Schnur R, Corman M, Cooper B, Dee M, Coty J (1995) erbB-2 oncogene inhibition by geldanamycin derivatives: synthesis, mechanism of action, and structure-activity relationships. J Med Chem 38:3813Google Scholar
  9. 9.
    Eiseman JL, Grimm A, Sentz DL, Lesser T, Gessner R, Zuhowski E, Nimieboka M, Egorin MJ (1999) Pharmacokinetics of 17-allylamino(17-demethoxy)geldanamycin in SCID mice bearing MDA.MB-453 xenografts and alterations in the expression of p185erb-B2 in the xenografts following treatment. Clin Cancer Res 5:3837sGoogle Scholar
  10. 10.
    Egorin MJ, Lagattuta TF, Hambruger DR, Covey JM, White KD, Musser SM, Eiseman JL (2002) Pharmacokinetics, tissue distribution, and metabolism of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (NSC 707545) in CD2F1 mice and Fischer 344 rats. Cancer Chemother Pharmacol 49:7Google Scholar
  11. 11.
    Fiebig H, Berger D, Dengler W, Wallbrecher E, Winterhalter B (1992) Combined in vitro/in vivo test procedure with human tumor xenografts. In: Fiebig HH, Berger D (eds) Immunodeficient mice in oncology. Karger Verlag, Basel, pp 321Google Scholar
  12. 12.
    Fiebig HH, Maier A, Burger AM (2004) Clonogenic assay with established human tumor xenografts: correlation of in vitro to in vivo activity as a basis for anticancer drug discovery. Eur J Cancer 40:802Google Scholar
  13. 13.
    Roth T, Burger AM, Dengler W, Fiebig HH (1999) Human tumor cell lines demonstrating the characteristics of patient tumors as useful models for anticancer drug development. In: Fiebig HH, Burger AM (eds) Relevance of tumor models for anticancer drug development. Karger Verlag, Basel, p 145Google Scholar
  14. 14.
    Hamburger A, Salmon S (1977) Primary bioassay of human tumor stem cells. Science 197:461Google Scholar
  15. 15.
    Alley M, Uhl C, Lieber, M (1982) Improved detection of drug cytotoxicity in the soft agar colony formation assay through use of a metabolizable tetrazolium salt. Life Sci 27:3071Google Scholar
  16. 16.
    Phillips RM, Burger AM, Loadman PM, Jarrett CM, Swaine DJ, Fiebig HH (2000) Predicting tumour responses to mitomycin C on the basis of DT-diaphorase activity or drug metabolism by tumour homogenates: implications for enzyme directed bioreductive drug development. Cancer Res 60:6384Google Scholar
  17. 17.
    Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesh H, Kenney S, Boyett JM (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82:1107Google Scholar
  18. 18.
    Brose MS, Volpe P, Feldman M, Kumar M, Rishi I, Gerrero R, Einhorn E, Herlyn M, Minna J, Nicholson A, Roth JA, Albelda SM, Davies H, Cox C, Brignell G, Stephens P, Futreal AP, Wooster R, Stratton MR, Weber BL (2002) BRAF and RAS mutations in human lung cancer and melanoma. Cancer Res 62:6997–7000Google Scholar
  19. 19.
    Burger AM, Fiebig HH, Stinson SF, Sausville EA (2004) 17-(allylamino)-17-demethoxy-geldanamycin activity in human melanoma models. Anticancer Drugs 15:377Google Scholar
  20. 20.
    Paull, KD, Shoemaker RH, Hodes L, Monks A, Scudiero DA, Rubinstein L, Plowman J, Boyd MR (1989) Display and analysis of patterns of differential activity of drugs against human tumor cell lines: development of mean graph and COMPARE algorithm. J Natl Cancer Inst 81:1088Google Scholar
  21. 21.
    Münster P, Marchion D, Basso A, Rosen N (2002) Degradation of HER2 by ansamycins induces growth arrest and apoptosis in cells with HER2 overexpression via a HER3, phosphatidylinositol 3′-kinase-AKT-dependent pathway. Cancer Res 62:3132Google Scholar
  22. 22.
    Calabrese C, Frank A, Maclean K, Gilbertson R (2003) Medulloblastoma sensitivity to 17-allylamino-17-demethoxygeldanamycin requires MEK/ERK. J Biol Chem 278:24951Google Scholar
  23. 23.
    Basso AD, Solit DB, Munster PN, Rosen N (2002) Ansamycin antibiotics inhibit Akt activation and cyclin D expression in breast cancer cells that overexpress HER2. Oncogene 21:1159Google Scholar
  24. 24.
    Clarke PA, Hostein I, Banerji U, Di Stefano F, Maloney A, Walton M, Judson I, Workman P (2000) 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 19:4125Google Scholar
  25. 25.
    Nimmanapalli, R, O’Bryan E, Bhalla K (2001) Geldanamycin and its analogue 17-allylamino-17-demethoxygeldanamycin lowers Bcr-Abl levels and induces apoptosis and differentiation of Bcr-Abl-positive human leukemic blasts. Cancer Res 61:1799Google Scholar
  26. 26.
    Solit DB, Zheng FF, Drobnjak M, Munster PN, Higgins B, Verbel D, Heller G, Tong W, Cordon-Cardo C, Agus DB, Scher HI, Rosen N (2002) 17-Allylamino-17-demethoxygeldanamycin induces the degradation of androgen receptor and HER-2/neu and inhibits the growth of prostate cancer xenografts. Clin Cancer Res 8:986Google Scholar
  27. 27.
    Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V, Menzies A, Mould C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake H, Gusterson BA, Cooper C, Shipley J, Hargrave D, Pritchard-Jones K, Maitland N, Chenevix-Trench G, Riggins GJ, Bigner DD, Palmieri G, Cossu A, Flanagan A, Nicholson A, Ho WCA, Leung SY, Yuen ST, Weber BL, Seigler HF, Darrow TL, Paterson H, Marais T, Marshall CJ, Wooster T, Stratton MR, Futreal PA (2002) Mutations of the BRAF gene is human cancer. Nature 417:949Google Scholar
  28. 28.
    Banerji U, Judson I, Workman P (2003) The clinical applications of heat shock protein inhibitors in cancer—present and future. Curr Cancer Drug Targets 3:385Google Scholar
  29. 29.
    Ehrlichman C, Toft D, Reid J, Goetz M, Ames M, Mandrekar S, Ajei A, McCollum A, Ivy P (2004) A phase I trial of 17-allylamino-geldanamycin (17-AAG) in patients with advanced cancer. J Clin Oncol ASCO Annual Meeting Proc 22(14S):202Google Scholar
  30. 30.
    Xu W, Marc M, Yuan X, Minnaugh E, Patterson C, Neckers L (2002) Chaperone-dependent E3 ubiquitin ligase CHIP mediates a degradative pathway for c-ErbB2/Neu. Proc Natl Acad Sci U S A 99:12847Google Scholar
  31. 31.
    Blank M, Mandel M, Keisari Y, Meruelo D, Lavie G (2003) Enhanced ubiquitinylation of heat shock protein 90 as a potential mechanism for mitotic cell death in cancer cells induced with hypericin. Cancer Res 63:8241Google Scholar
  32. 32.
    Smith V, Hobbs S, Court W, Eccles S, Workman P, Kelland LR (2002) ErbB2 overexpression in an ovarian cancer cell line confers sensitivity to the Hsp90 inhibitor geldanamycin. Anticancer Res 22:1993Google Scholar
  33. 33.
    Gorden A, Osman I, Gai W, He D, Huang W, Davidson A, Houghton AN, Busam K, Polsky D (2003) Analysis of BRAF and N-RAS mutations in metastatic melanoma tissues. Cancer Res 63:3955Google Scholar
  34. 34.
    Grbovic OM, Basso AD, Friedlander P, Houghton A, Solit DB, Rosen N (2004) Activate, mutated B-raf protein kinase requires the Hsp90 chaperone for folding and stability and is degraded in response to Hsp90 inhibitors (abstract 100). Proc Am Assoc Cancer Res 45Google Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Victoria Smith
    • 1
  • Edward A. Sausville
    • 2
    • 3
  • Richard F. Camalier
    • 2
  • Heinz-Herbert Fiebig
    • 1
  • Angelika M. Burger
    • 1
    • 4
  1. 1.Tumor Biology Center at the University of FreiburgFreiburgGermany
  2. 2.Developmental Therapeutics ProgramDCTD, NCIBethesdaUSA
  3. 3.Associate Director for Clinical Research, University of Maryland Greenebaum Cancer CenterUniversity of Maryland MedicineBaltimoreUSA
  4. 4.Sunnybrook & Women’s College Health Sciences CentreTorontoCanada

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