Lactandrate: a D-homo-aza-androsterone alkylator in the treatment of breast cancer

  • Dimitrios T.P. Trafalis
  • George D. Geromichalos
  • Catherine Koukoulitsa
  • Athanasios Papageorgiou
  • Panayiotis Karamanakos
  • and Charalambos Camoutsis
Preclinical study

Summary

The sensitivity of breast neoplasms to hormonal control provides the basis of novel investigational treatments with steroidal alkylators. An androsterone D-lactam steroidal ester, the 3β-hydroxy-13α-amino-13,17-seco- 5α-androstan-17-oic-13,17-lactam, p-bis(2-chloroethyl)amino phenyl acetate (lactandrate) was synthesized and tested for antitumor activity against six human breast cancer cell lines in vitro and against two murine and one xenograft mammary tumors in vivo. A docking study on the binding interactions of lactandrate with the ligand-binding domain (LBD) of estrogen receptor-alpha (ERα) was inquired. In vitro testing of lactandrate cytostatic and cytotoxic activity was performed on T47D, MCF7, MDA-MB-231, BT-549, Hs578T, MDA-MB-435 breast adenocarcinoma human cell lines. In vivo testing was performed on two murine mammary tumors, the MXT tumor and CD8F1 adenocarcinoma, as well as on human mammary carcinoma MX-1 xenograft. Molecular modeling techniques were adopted to predict a possible location and interaction mode of the molecule into LBD. Lactandrate induced significantly high antitumor effect against all tested in vitro and in vivo models. The cell lines with positive ER expression found to be significantly more sensitive to lactandrate. Moreover, lactandrate found to be positioned inside the binding cavity with its steroidal moiety, whilst the alkylating moiety protrudes out of receptor’s pocket. Lactandrate produced important anticancer activity on breast cancer in vitro and in vivo. Some correlation between ER and lactandrate effect was demonstrated. Docking studies provide the basis for the structure-based design of improved steroidal alkylating esters for the treatment of estrogen-related cancers.

Keywords

breast cancer docking estrogen receptor homo-aza-steroids human cell lines human breast xenograft lactam murine breast tumors steroid alkylators 

Abbreviations

AF-2

activation function 2

AWC

average mice weight change

CR

complete tumor regression rate

DMSO

dimethyl sulfoxide

ER

estrogen receptor

GI50

50% growth inhibition

hERα

human estrogen receptor alpha

IC50

50% in vitro cytotoxicity

i.p.

intra-peritoneal

LBD

ligand-binding domain

MTD

maximum tolerated dose

PKC

protein kinase C

PR

partial tumor regression rate

TGI

total growth inhibition

TFS

tumor-free survivors

TI

tumor inhibition

TW

tumor weight

%T/C

% median lifespan change of treated animals (T) over the control (C)

References

  1. 1.
    Wong ZW, Ellis MJ, First-line endocrine treatment of breast cancer: aromatase inhibitor or antioestrogen? Br J Cancer 90: 20–25, 2004CrossRefPubMedGoogle Scholar
  2. 2.
    Mouridsen HT, Rose C, Brodie AH, Smith IE, Challenges in the endocrine management of breast cancer Breast 2(Suppl): S2–19, 2003CrossRefGoogle Scholar
  3. 3.
    Catsoulacos P, Politis D, Wampler GL, A new steroidal alkylating agent with improved activity in advanced murine leukemias Cancer Chemother Pharmacol 3: 67–70, 1979CrossRefPubMedGoogle Scholar
  4. 4.
    Catsoulacos P, Camoutsis C, Wampler GL, Effect of a Δ5-homo-aza-steroidal ester in P388 and L1210 murine leukemias Oncology 39: 59–60, 1982PubMedGoogle Scholar
  5. 5.
    Catsoulacos P, Activity of 3β-hydroxy-13α-amino-13,17-seco-5α-androstan-17-oic-13, 17-lactam p-bis (2-chloroethyl)aminophenoxy acetate (NSC-294859) on experimental tumor and leukemia systems. Oncology 40: 290–292, 1983PubMedCrossRefGoogle Scholar
  6. 6.
    Catsoulacos P, Wampler GL, Activity of 3β-hydroxy-13α-amino-13,17-seco-5α-androstan-17-oic-13,17-lactam(p-bis(2-chloroethyl)amino)-phenyl)acetate (NSC-290205) in murine solid tumors Oncology 39: 109–112, 1982PubMedGoogle Scholar
  7. 7.
    Catsoulacos P, Further studies on the anti-neoplastic activity of 3β-hydroxy-13α-amino-13,17-seco-5α-androstan-17-oic-13,17-lactam(p-bis(2-chloroethyl)amino)phenyl)acetate (NSC-290205). Cancer Lett 22: 199–202, 1984CrossRefPubMedGoogle Scholar
  8. 8.
    Wall ME, Abernethy SG, Carroll JFI, Taylor DJ, The effects of some steroidal alkylating agents on experimental animal mammary tumor and leukemia systems J Med Chem 12: 810–818, 1969CrossRefPubMedGoogle Scholar
  9. 9.
    Wampler GL, Catsoulacos P, Antileukemic effect of homo-aza-steroidal-ester of [p-[bis(2-chloroethyl)amino]acetic acid Cancer Treat Rep 61: 37–41, 1977PubMedGoogle Scholar
  10. 10.
    Tsai M-J, O’Malley BW, Molecular mechanisms of action of steroid/thyroid receptor superfamily members Annu Rev Biochem 63: 451–486, 1994CrossRefPubMedGoogle Scholar
  11. 11.
    Beato M, Sanchez-Pacheco A, Interaction of steroid hormone receptors with the transcription initiation complex Endocr Rev 17: 587–609, 1996CrossRefPubMedGoogle Scholar
  12. 12.
    Shiau AK, Barstad D, Loria M, Cheng L, Kushner PJ, Agard DA, Greene GL ,The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen Cell 95: 927–937, 1998Google Scholar
  13. 13.
    Catsoulacos P, Boutis L, Aza-steroids. Beckmann rearrangement of 3β-acetoxy-5α-androstan-17-one oxime acetate with boron fluoride. Alkylting agents. Chim Ther 8: 215–217, 1973Google Scholar
  14. 14.
    Finlay GJ, Wilson WR, Baguley BC, Comparison of in vitro activity of cytotoxic drugs towards human carcinoma and leukaemia cell lines Eur J Cancer Clin Oncol 22: 655–662, 1986CrossRefPubMedGoogle Scholar
  15. 15.
    Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine DL, Abbott BJ, Mayo JG, Shoemaker RH, Boyd MR, Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay Cancer Res, 48: 589–601, 1988Google Scholar
  16. 16.
    Boyd MR, Paull KD, Some practical considerations and applications of the National Cancer Institute in vitro anticancer drug discovery screen Drug Devel Res 34: 91–109, 1995CrossRefGoogle Scholar
  17. 17.
    Francisco J, Pauwels Ο, Simon S, Gasperin P, Van Houtte P, Pasteels JL, Kiss R, Computer-assisted morphonuclear characterization of radiotherapy-induced effects in MXT mouse mammary adenocarcinomas surviving earlier radiotherapy Int J Radiat Oncol Biol Phys 32: 409–419, 1995CrossRefPubMedGoogle Scholar
  18. 18.
    Danguy A, Kiss R, Leclercq G, Heuson JC, Pasteels JL, Morphology of MXT mouse mammary tumors. Correlation with growth characteristics and hormone sensitivity Eur J Cancer Clin Oncol 22: 69–76, 1986CrossRefPubMedGoogle Scholar
  19. 19.
    Martin DS, Fugmann RA, Stolfi RL, Hayworth PE, Solid tumor animal model therapeutically predictive for human breast cancer Cancer Chemother Rep 5: 89–109, 1975Google Scholar
  20. 20.
    Goldin A, Vendi JM, MacDonald JS, Muggia FM, Henney JE, DeVita VT Jr, Current results of the screening program at the Division of Cancer Treatment, National Cancer Institute Eur J Cancer, 17: 129–142, 1981PubMedGoogle Scholar
  21. 21.
    Stolfi RL, Martin DS, Fugmann RA, Spontaneous mutine mammary adenocarcinoma: model system for evaluation of combined methods of therapy Cancer Chemother Rep 55: 239–251, 1971. PubMedGoogle Scholar
  22. 22.
    NIH Principles of laboratory animal care. NIH publication 85: 23, 1985Google Scholar
  23. 23.
    Workman P, Twentyman P, Balkwill F, Balmain A, Chaplin D, Double J, Embleton J, Newell D, Raymond R, Stables J, Stephens T, Wallace J, United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR) guidelines for the welfare of animals in experimental neoplasia (second edition) Br J Cancer 77: 1–10, 1998Google Scholar
  24. 24.
    Carosati E, Sciabola S, Gabriele C, Hydrogen bonding interactions of covalently bonded fluorine atoms: from crystallographic data to a new angular function in the GRID force field J Med Chem 47: 5114–5125, 2004CrossRefPubMedGoogle Scholar
  25. 25.
    Goodford PJ, 1985 A computational procedure for determining energetically favourable binding sites on biologically important macromolecules J Med Chem 28: 849–857CrossRefPubMedGoogle Scholar
  26. 26.
    Brozowski AM, Pike ACW, Dauter Z, Hubbard RE, Bonn T, Engström O, Öhman L, Greene GL, Gustafsson JÅ, Carlquist M, 1997 Molecular basis of agonism and antagonism in the oestrogen receptor Nature 389: 753–758CrossRefGoogle Scholar
  27. 27.
    Boyer S, Zamora I, New methods in predicting metabolism J Comput-Aided Mol Des 16: 403–413, 2002CrossRefPubMedGoogle Scholar
  28. 28.
    Zamora I, Afzelius L, Cruciani G, Predicting drug metabolism: a site of metabolism prediction tool applied to the cytochrome P450 2C9 J Med Chem 46: 2313–2324, 2003CrossRefPubMedGoogle Scholar
  29. 29.
    Boobis A, Gundert-Remy U, Kremers P, Macheras P, Pelkonen O, In silico prediction of ADME and pharmacokinetics. Report of an expert meeting organised by COST B15 Eur J Pharm Sci 17: 183–193, 2002CrossRefPubMedGoogle Scholar
  30. 30.
    Wrighton SA, Schuetz EG, Thummel KE, Shen DD, Korzekwa KR, Watkins PB, The human CYP3A subfamily: practical considerations Drug Metab Rev 32: 339–361, 2000CrossRefPubMedGoogle Scholar
  31. 31.
    Guengerich FP, 2001 Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity Chem Res Toxicol 14: 611–650CrossRefPubMedGoogle Scholar
  32. 32.
    Crivori P, Zamora I, Speed B, Orrenius C, Poggesi I, Model based on GRID-derived descriptors for estimating CYP3A4 enzyme stability of potential drug candidates J Comput-Aid Mol Des 18: 155–166, 2004CrossRefGoogle Scholar
  33. 33.
    Renaud J, Bischoff SF, Buhl T, Floersheim P, Fournier B, Halleux C, Kallen J, Keller H, Schlaeppi J-M, Stark W, Estrogen receptor modulators: identification and structure-relationships of potent ER-selective tetrahydroisoquinoline ligands J Med Chem 46: 2945–2957, 2003CrossRefPubMedGoogle Scholar
  34. 34.
    Kekenes-Huskey PM, Muegge I, Rauch M, Gust R, Knapp E-W, A molecular docking study of estrogenically active compounds with 1,2-diarylethane and 1,2-diarylethene pharmacophores Bioorg Med Chem 12: 6527–6537, 2004CrossRefPubMedGoogle Scholar
  35. 35.
    Jones KL, Buzdar AU, Jones KL, Buzdar AU, A review of adjuvant hormonal therapy in breast cancer Endocr Relat Cancer 11: 391–406, 2004CrossRefPubMedGoogle Scholar
  36. 36.
    Leclercq G, Devleeschouwer N, Pairas G, Camoutsis C, Catsoulacos P, Effect of an homo-aza-steroid on estrogen receptor Meth Find Exp Clin Pharmacol 5: 365–367, 1983Google Scholar
  37. 37.
    Cho W-J, Min SY, Le TN, Kim TS, Synthesis of new 3-arylsoquinolinamines: effect on topoisomerase I inhibition and cytotoxicity Bioorg Med Chem Lett 13: 4451–4454, 2003CrossRefPubMedGoogle Scholar
  38. 38.
    De la Fuente JA, Manzanaro S, Martin MJ, de Quesada TG, Reymundo I, Luengo SM, Gago F, Synthesis, activity, and molecular modeling studies of novel human aldose reductase inhibitors based on a marine natural product J Med Chem 46: 5208–5221, 2003CrossRefGoogle Scholar
  39. 39.
    Sarno S, de Moliner E, Ruzzene M, Pagano MA, Battistuta R, Bain J, Fabbro D, Schoepfer J, Elliott M, Furet P, Meggio F, Zanotti G, Pinna LA, Biochemical and three-dimensional-structural study of the specific of protein kinase CK2 by [5-oxo-5,6-dihydroindolo-(1,2-a)quinazolin-7-yl]acetic acid (IQA) Biochem J 374: 639–646, 2003CrossRefPubMedGoogle Scholar
  40. 40.
    Tzukerman MT, Esty A, Santiso-Mere D, Danelian D, Parker MG, Stein RB, Pike JW, McDonnell DP, Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions Mol Endocrinol 8: 21–30, 1994CrossRefPubMedGoogle Scholar
  41. 41.
    Kabara JJ, Holzschu DL, Catsoulacos P, Structure function activity of azasterols and nitrogen containing steroids Lipids 11: 7555–7562, 1976CrossRefGoogle Scholar
  42. 42.
    Tsuchiya Y, Nakajima M, Yokoi T, Cytochrome P450-mediated metabolism of estrogens and its regulation in human Cancer Lett 20: 1–10, 2004Google Scholar
  43. 43.
    Torimoto N, Ishii I, Hata M, Nakamura H, Imada H, Ariyoshi N, Ohmori S, Igarashi T, Kitada M, 2003 Direct interaction between substrates and endogenous steroids in the active site may change the activity of cytochrome P450 3A4 Biochemistry 42: 15068–15077CrossRefPubMedGoogle Scholar
  44. 44.
    Szklarz GD, Halpert JR, 1997 Molecular modeling of cytochrome P450 3A4 J Comput Aided Mol Des 1: 265–272CrossRefGoogle Scholar
  45. 45.
    Papageorgiou A, Ivanov IG, Markov GG, Koliais SI, Boutis L, Catsoulacos P, Interaction of homo-aza-steroidal ester of [p-[bis-(2-chloroethyl) amino] acetic acid (ASE) with DNA of Ehrlich ascites tumor cells FEBS Lett 153: 194–198, 1983CrossRefPubMedGoogle Scholar
  46. 46.
    Shepherd RE, Huff K, McGuire WL, Brief communication: estrogen receptor interaction with antitumor agent estradiol mustard J Nat Cancer Inst 53: 895–897, 1974PubMedGoogle Scholar
  47. 47.
    Kubota T, Kawamura E, Suzuki T, Yamada T, Toyoda H, Miyagawa T, Kurokawa T,Antitumor activity and pharmacokinetics of estra-1,3,5 (10)-triene-3,17 beta-diol, 3-benzoate, 17-((4-(4-bis(2-chloroethyl)amino)phenyl)-1-oxobutoxy) acetate) (Bestrabucil) in human tumor xenografts serially transplanted into nude mice Jpn J Clin Oncol 16: 357–564, 1986PubMedGoogle Scholar
  48. 48.
    Catsoulacos P, Catsoulacos D, Antitumor activity of homo-aza-steroidal esters of p-N,N-bis(2-chloroethyl) amino phenoxy acetic acid Anticancer Res 13: 1203–1208, 1993PubMedGoogle Scholar
  49. 49.
    Camoutsis C, Trafalis DTP, An overview on the antileukemic potential of D-homo-aza- and respective 17β-acetamido-steroidal alkylating esters Invest New Drugs 21: 47–54, 2003CrossRefPubMedGoogle Scholar
  50. 50.
    Trafalis DTP, Camoutsis C, Dalezis P, Papageorgiou A, Kontos M, Karamanakos P, Giannakos G, Athanassiou AE, Antitumour effect of A- and D-lactam androgen nitrogen mustards on non-small cell lung carcinoma J BUON 9: 75–82, 2004Google Scholar
  51. 51.
    Ma D, Wang G, Wang S, Kozikowski AP, Lewin NE, Blumberg PM, Synthesis and protein kinase C binding activity of benzolactam-V-7 Bioorg Med Chem Lett 9: 1371–1374, 1999CrossRefPubMedGoogle Scholar
  52. 52.
    Endo Y, Shimazu M, Fukasawa H, Driedger PE, Kimura K, Tomioka N, Itai A, Shudo K, Synthesis computer modelling and biological evaluation of novel protein kinase C agonists based on a 7-membered lactam moiety Bioorg Med Chem Lett 9: 173–178, 1999CrossRefPubMedGoogle Scholar
  53. 53.
    Endo Y, Yokohama A, Role of the hydrophobic moiety of tumor promoters. Synthesis and activity of 2-alkylated benzolactams Bioorg Med Chem Lett 10: 63–66, 2000CrossRefPubMedGoogle Scholar
  54. 54.
    Trafalis DTP, Camoutsis C, Karamanakos P, Arvanitis A, Tegou E, Ziras N, Athanassiou AE, Preclinical evaluation of the homo-aza-steroidal ester: 13β-hydroxy-13α-amino-13,17-seco-5α-androstan-17-oic-13,17-lactam-p-bis(2-chloro ethyl) aminophenoxy acetate for the treatment of malignant melanoma J BUON 8: 333–339, 2003PubMedGoogle Scholar
  55. 55.
    Catsoulacos P, Politis D, Wampler GL, Antitumor activity of homo-aza-steroidal esters of [p-bis(2-chloroethyl)amino]phenyl]acetic acid [p-bis(2-chloroethyl)amino] phenyl]butyric acid Cancer Chemother Pharmacol 10: 129–132, 1983CrossRefPubMedGoogle Scholar
  56. 56.
    Catsoulacos P, Camoutsis C, Papageorgiou A, Adamiak-Margariti E, Cytostatic effect of homo-aza-steroidal esters in vivo and in vitro structure–activity relationships Anticancer Res 12: 1617–1620, 1992PubMedGoogle Scholar
  57. 57.
    Dalmases P, Gomez-Belinchon JI, Bonet J-J, Giner-Sorolla A, Schmid FA, Antineoplastic agents II. A nitrogen mustard derivative of N-methylated steroidal lactam Eur J Med Chem 18: 541–543, 1983Google Scholar
  58. 58.
    Dalmases P, Cervantes G, Quintana J, Bonet J-J, Antineoplastic agents. V. Nitrogen mustards of systematically modified steroidal ring A lactams Eur J Med Chem 19, 465–467, 1984Google Scholar
  59. 59.
    Catsoulacos P, Papageorgiou A, Margariti E, Mourelatos D, Mioglou E, Comparison of current alkylating agents with a homo-aza-steroidal ester for antineoplastic activity Oncology 51: 74–78, 1994PubMedGoogle Scholar
  60. 60.
    Tiersten AD, Nelsen C, Talbot S, Vahdat L, Fine R, Troxel A, Brafman L, Shriberg L, Antman K, Petrylak DP, A phase II trial of docetaxel and estramustine in patients with refractory metastatic breast carcinoma Cancer 97: 537–544, 2003. CrossRefPubMedGoogle Scholar
  61. 61.
    Ζelek L, Βarthier S, Riofrio M, Sevin D, Fizazi K, Spielmann M, Single-agent estramustine phosphate (EMP) is active in advanced breast cancer after failure with anthracyclines and taxanes Ann Oncol 12: 1265–1268, 2001CrossRefGoogle Scholar
  62. 62.
    Kubota T, Abe O, Izuo M, Watanabe H, Enomoto K, Ohsawa N, Kuno K, A phase II multi-institutional study of estra-1,3,5(10)-triene-3,17 beta-diol, 3-benzoate, 17[[4-[4-[bis(2-chloroethyl)amino]phenyl]-1- oxobutoxy]acetate] (KM2210), a novel antitumor agent, for advanced and recurrent breast carcinoma Anticancer Res 13: 2361–2365, 1993PubMedGoogle Scholar
  63. 63.
    Kubota T, Ishibiki K, Abe O, Kosano H, Ohsawa N, Hoffman RM, Mode of action of estra-1,3,5(10)-triene-3, 17 beta-diol, 3-benzoate, 17[4-[4-[bis(2-chloroethyl) amino]phenyl]-1-oxobutoxy] acetate] (KM2210) on MCF-7 human breast tumours transplanted into nude mice Anticancer Res 13: 935–940, 1993PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  • Dimitrios T.P. Trafalis
    • 1
    • 2
    • 6
  • George D. Geromichalos
    • 3
  • Catherine Koukoulitsa
    • 4
  • Athanasios Papageorgiou
    • 3
  • Panayiotis Karamanakos
    • 5
  • and Charalambos Camoutsis
    • 1
  1. 1.Laboratory of Medicinal Chemistry, Faculty of PharmacyUniversity of PatrasPatrasGreece
  2. 2.1st Department of Medical Oncology“Metaxa” Cancer HospitalPiraeusGreece
  3. 3.Symeonidio Research CenterTheagenio Cancer HospitalThessalonikiGreece
  4. 4.Department of Pharmacognosy and Chemistry of Natural Products, School of PharmacyUniversity of AthensAthensGreece
  5. 5.1st Department of SurgeryMedical School, University of AthensAthensGreece
  6. 6.AthensGreece

Personalised recommendations