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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


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.

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Phosphate-buffered saline


National Cancer Institute




Heat shock protein


Melanoma xenograft established by Fiebig et al.


Poly-adenosine ribose polymerase


Total growth inhibition (no change vs initial cell number)


Growth-inhibitory concentration 50% compared to control


Tumor clonogenic assay


Ethylenediaminetetraacetic acid


  1. Maloney A, Workman P (2002) HSP90 as a new therapeutic target for cancer therapy: the story unfolds. Expert Opin Biol Ther 2:3

    Google Scholar 

  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 61:4003

    Google Scholar 

  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 277:39858

    Google Scholar 

  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 277:10346

    Google Scholar 

  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 89:239

    Google Scholar 

  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 90:65

    Google Scholar 

  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 38:308

    Google Scholar 

  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 38:3813

    Google Scholar 

  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 5:3837s

    Google Scholar 

  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 49:7

    Google Scholar 

  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 321

    Google Scholar 

  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 40:802

    Google Scholar 

  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 145

    Google Scholar 

  14. Hamburger A, Salmon S (1977) Primary bioassay of human tumor stem cells. Science 197:461

    Google Scholar 

  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 27:3071

    Google Scholar 

  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 60:6384

    Google Scholar 

  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 82:1107

    Google Scholar 

  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 62:6997–7000

    Google Scholar 

  19. Burger AM, Fiebig HH, Stinson SF, Sausville EA (2004) 17-(allylamino)-17-demethoxy-geldanamycin activity in human melanoma models. Anticancer Drugs 15:377

    Google Scholar 

  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 81:1088

    Google Scholar 

  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 62:3132

    Google Scholar 

  22. Calabrese C, Frank A, Maclean K, Gilbertson R (2003) Medulloblastoma sensitivity to 17-allylamino-17-demethoxygeldanamycin requires MEK/ERK. J Biol Chem 278:24951

    Google Scholar 

  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 21:1159

    Google Scholar 

  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 19:4125

    Google Scholar 

  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 61:1799

    Google Scholar 

  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 8:986

    Google Scholar 

  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 417:949

    Google Scholar 

  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 3:385

    Google Scholar 

  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):202

    Google Scholar 

  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 99:12847

    Google Scholar 

  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 63:8241

    Google Scholar 

  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 22:1993

    Google Scholar 

  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 63:3955

    Google Scholar 

  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 45

    Google Scholar 

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This work was supported by a contract from the US NCI (no. N01-CM-27026) to A.M.B. and H.H.F. We wish to thank Dr. Armin Maier and Anke Masch for their assistance with performing and evaluating the clonogenic assays and Dr. Aiguo Zhang for her help with the BRAF mutation analyses.

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Correspondence to Edward A. Sausville.

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Smith, V., Sausville, E.A., Camalier, R.F. et al. 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. Cancer Chemother Pharmacol 56, 126–137 (2005).

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  • 17DMAG
  • 17AAG
  • Hsp90 modulation
  • Melanoma