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Molecular and Cellular Biochemistry

, Volume 357, Issue 1–2, pp 343–352 | Cite as

2-Methoxyestradiol-bis-sulfamate induces apoptosis and autophagy in a tumorigenic breast epithelial cell line

  • M. H. Visagie
  • A. M. Joubert
Article

Abstract

In anticancer research where the focus is on finding agents that induces cell death while leaving non-tumorigenic cells less affected, a novel 2-methoxyestradiol derivative has come forth. 2-Methoxyestradiol-bis-sulfamate (2-MeOE2bisMATE) is a 2-methoxyestradiol derivative produced by bis-sulphamoylation, which possesses increased antiproliferative activity and biological availability. Several questions remain regarding the type of cell death mechanisms and possible induction of autophagy by 2-MeOE2bisMATE. The aim of this in vitro study was to investigate the cell death mechanisms exerted by 2-MeOE2bisMATE in an adenocarcinoma cell line (MCF-7) by analyzing its influence on cell growth, morphology, and possible induction of cell death. Spectrophotometry (crystal violet staining), transmission electron microscopy (TEM), light microscopy (hematoxylin and eosin staining), and fluorescent microscopy (Hoechst 33342, propidium iodide and acridine orange) were employed. Spectrophotometrical studies indicated that 2-MeOE2bisMATE decreased cell numbers to 75% in MCF-7 cells after 24 h and to 47% after 48 h of exposure. TEM demonstrated membrane blebbing, nuclear fragmentation, and chromatin condensation indicating the hallmarks of apoptosis. Light microscopy revealed the presence of several cells blocked in metaphase, and apoptotic cells were also observed. Fluorescent microscopy demonstrated increased lysosomal staining; suggesting the induction of autophagy. 2-MeOE2bisMATE shows therapeutic potential, as an, anticancer agent, and the investigation of the cell death mechanisms used by 2-MeOE2bisMATE, thus, warrants further investigation.

Keywords

2-Methoxyestradiol-bis-sulfamate Apoptosis Autophagy MCF-7 

Notes

Acknowledgments

This study was supported by grants from the Medical Research Council of South Africa, the Cancer Association of South Africa, RESCOM (School of Medicine, University of Pretoria, South Africa), National Research Foundation, and the Struwig-Germeshuysen Cancer Research Trust of South Africa.

References

  1. 1.
    Banerjeei SK, Zoubine MN, Sarkar DK, Weston AP, Shah JH, Campbell DR (2000) 2-Methoxyestradiol blocks estrogen-induced rat pituitary tumor growth and tumor angiogenesis: possible role of vascular endothelial growth factor. Anticancer Res 20:2641–2645PubMedGoogle Scholar
  2. 2.
    Pribluda VS, Gubish ER, La Vallee TM, Treston A, Swartz GM, Green SJ (2000) 2-Methylestradiol: an endogenous antiangiogenic and antiproliferative drug candidate. Cancer Metastasis Rev 19:173–179PubMedCrossRefGoogle Scholar
  3. 3.
    Lavallee TM, Zhan XH, Herbstritt CJ, Kough EC, Green SJ, Pribluda VS (2002) 2-Methoxyestradiol inhibits proliferation and induces apoptosis independently of estrogen receptors alpha and beta. Cancer Res 62:3691–3697PubMedGoogle Scholar
  4. 4.
    Lippert TH, Adlercreutz H, Berger MR, Seeger H, Elger W, Mueck AO (2003) Effect of 2-methoxyestradiol on the growth of methyl-nitroso-urea (MNU)-induced rat mammary carcinoma. J Steroid Biochem Mol Biol 84:51–56PubMedCrossRefGoogle Scholar
  5. 5.
    Bhati R, Gokmen-Polar Y, Sledge GW, Fan C, Nakshatri H, Ketelsen D, Borchers CH, Dial MJ, Patterson C, Klauber-DeMore N (2007) 2-Methoxyestradiol inhibits the anaphase-promoting complex and protein translation in human breast cancer cells. Cancer Res 67:702–708PubMedCrossRefGoogle Scholar
  6. 6.
    Funakoshi T, Birsner AE, D’Amato RJ (2006) Antiangiogenic effect of oral 2-methoxyestradiol on choroidal neovascularization in mice. Exp Eye Res 83:1102–1107PubMedCrossRefGoogle Scholar
  7. 7.
    Matei D, Schilder J, Sutton G, Perkins S, Breen T, Quon C, Sidor C (2009) Activity of 2 methoxyestradiol (Panzem® NCD) in advanced, platinum-resistant ovarian cancer and primary peritoneal carcinomatosis: a hoosier oncology group trial. Gynaecol Oncol 115:90–96CrossRefGoogle Scholar
  8. 8.
    Lakka SS, Rao JS (2008) Angiogenic therapy in brain tumors. Expert Rev Neurother 8:1457–1473PubMedCrossRefGoogle Scholar
  9. 9.
    Sutherland TE, Anderson RL, Hughes RA, Altmann E, Schuliga M, Ziogas J, Stewart AG (2007) 2-Methoxyestradiol—a unique blend of activities generating a new class of anti-tumour/anti-inflammatory agents. Drug Discov Today 12:577–584PubMedCrossRefGoogle Scholar
  10. 10.
    Lakhani NJ, Lepper ER, Sparreboom A, Dahut WL, Venitz J, Figg WD (2005) Determination of 2-methoxyestradiol in human plasma, using liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 19:1176–1182PubMedCrossRefGoogle Scholar
  11. 11.
    Foster PA, Newman SP, Leese MP, Bernetiere S, Diolez C, Camara J, Hacher B, Baronnet MM, Ali T, Potter BV, Reed MJ, Purohit A (2008) A new micronized formulation of 2-methoxyestradiol-bis-sulphamate (STX140) is therapeutically potent against breast cancer. Anticancer Res 28:577–581PubMedGoogle Scholar
  12. 12.
    Suzuki RN, Newman SP, Purohit A, Leese MP, Potter BV, Reed MJ (2003) Growth inhibition of multi-drug-resistant breast cancer cells by 2-methoxyoestradiol-bis-sulphamate and 2-ethyloestradiol-bis-sulphamate. J Steroid Biochem Mol Biol 84:269–278PubMedCrossRefGoogle Scholar
  13. 13.
    Foster PA, Ho YT, Newman SP, Kasprzyk MP, Leese MP, Potter BV, Reed MJ, Purohi A (2007) 2-MeOE2bisMATE and 2-EtE2bisMATE induce cell cycle arrest and apoptosis in breast cancer xenografts as shown by a novel ex vivo technique. Breast Cancer Res Treat 111:251–260PubMedCrossRefGoogle Scholar
  14. 14.
    Newman SP, Foster PA, Stengel C, Day JM, HO YT, Judde JG, Lassalle M, Prevost G, Leese MP, Potter BV, Reed MJ, Purohit A (2008) STX140 is efficacious in vitro and in vivo taxane-resistant breast carcinoma cells. Clin Cancer Res 14:597–606PubMedCrossRefGoogle Scholar
  15. 15.
    Newman SP, Leese MP, Purohit A, James DRC, Rennie CE, Potter BVL (2004) Inhibition of in vitro angiogenesis by 2-methoxy- and 2-ethyl-estrogen sulfamates. Int J Cancer 109:533–540PubMedCrossRefGoogle Scholar
  16. 16.
    Joubert AM, Marais S, Maritz C (2009) Influence of 2-methoxyestradiol on MCF-7 cells: An improved differential interference contrasting technique and Bcl-2 and Bax protein expression levels. Biocell 33:67–70PubMedGoogle Scholar
  17. 17.
    Gillies RJ, Didier N, Denton M (1986) Determination of cell number in monolayer cultures. Anal Biochem 159:109–113PubMedCrossRefGoogle Scholar
  18. 18.
    Grever MR, Schepartz SA, Chabner BA (1992) The National Cancer Institute: cancer drug discovery and development program. Semin Oncol 19:622–638PubMedGoogle Scholar
  19. 19.
    Kusuzaki K, Murata H, Takeshita H, Hashiguchi S, Nozaki T, Emoto K, Ashihara T, Hirasawa Y (2000) Intracellular binding sites of acridine orange in living osteosarcoma cells. Anticancer Res 20:971–975PubMedGoogle Scholar
  20. 20.
    Klionsky DJ, Cuervo AM, Seglen PO (2007) Methods for monitoring autophagy from yeast to human. Autophagy 3:181–206PubMedGoogle Scholar
  21. 21.
    Kanzawa T, Germano IM, Komata T, Ito H, Kondo, Kondo S (2004) Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death Differ 11:448–457PubMedCrossRefGoogle Scholar
  22. 22.
    Elmore S (2007) A review of programmed cell death. Toxicol Pathol 35:495–516PubMedCrossRefGoogle Scholar
  23. 23.
    Newman SP, Foster PA, Ho YT, Day JM, Raobaikady B, Kasprzyk PG, Leese MP, Potter BV, Reed MJ, Purohit A (2007) The therapeutic potential of a series of bioavailability anti-angiogenic microtubule disruptors as therapy for hormone-independent prostate and breast cancers. Br J Cancer 97:1673–1682PubMedCrossRefGoogle Scholar
  24. 24.
    Utsumi T, Leese MP, Chander SK, Gaukroger K, Purohit A, Newman SP, Potter BVL, Reed MJ (2005) The effects of 2-methoxyoestrogen sulphamates on the in vitro and in vivo proliferation of breast cancer cells. J Steroid Biochem Mol Biol 94:219–227PubMedCrossRefGoogle Scholar
  25. 25.
    Day JM, Foster PA, Tutill HJ, Newman SP, Ho YT, Leese MP, Potter BM, Reed MJ, Purohit A (2009) BCRP expression does not result in resistance to STX140 in vivo, despite the increased expression of BCRP in A2780 cells in vitro after long-term STX140 exposure. Br J Cancer 100:476–486PubMedCrossRefGoogle Scholar
  26. 26.
    Foster PA, Ho YT, Newman SP, Leese MP, Potter BV, Reed MJR, Purohit A (2009) STX140 and STX641 Cause apoptosis via the intrinsic mitochondrial pathway and down-regulate Survivin and XIAP expression in ovarian and prostate cancer cells. Anticancer Res 29:3751–3757PubMedGoogle Scholar
  27. 27.
    Wood L, Leese MP, Mouzakiti A, Purohit A, Potter BV, Reed MJ, Packham G (2004) 2-MeOE2bisMATE induces caspase-dependent apoptosis in CAL51 breast cancer cells and overcomes resistance to TRAIL via cooperative activation of caspases. Apoptosis 9:323–332PubMedCrossRefGoogle Scholar
  28. 28.
    Jeong D, Kim TS, Lee JW, Kim KT, Kim HJ, Kim IH, Kim IY (2001) Blocking of acidosis-mediated apoptosis by a reduction of lactate dehydrogenase activity through antisense mRNA expression. Biochem Biophys Res Commun 289:1141–1149PubMedCrossRefGoogle Scholar
  29. 29.
    Xia GH, Chen BA, Shao ZY, Lu HX, Konstanze D, Hartmut D (2007) Mechanism of 2-methoxyestradiol-induced apoptosis in myelodysplastic syndrome MUTZ-1 cell line. Zhongguo Shi Yan Xue Ye Xue Za Zhi 15:296–301PubMedGoogle Scholar
  30. 30.
    Ireson CR, Chander SK, Purohit A, Perera S, Newman SP, Parish D, Leese MP, Smith AC, Reed MJ (2004) Pharmacokinetics and efficacy of 2-methoxyoestradiol and 2-methoxyoestradiol-bis-sulphamate in vivo in rodents. Br J Cancer 90:932–937PubMedCrossRefGoogle Scholar
  31. 31.
    Tsujimo Y, Shimizu S (2005) Another way to die: autophagic programmed cell death. Cell Death Differ 12:1528–1534CrossRefGoogle Scholar
  32. 32.
    Azad MB, Chen Y, Gibson SB (2009) Regulation of autophagy by reactive oxygen species (ROS): implication for cancer progression and treatment. Antioxid Redox Signal 11:1–14CrossRefGoogle Scholar
  33. 33.
    Nishida K, Yamaguchi O, Otsu K (2008) Crosstalk between autophagy and apoptosis ion heart disease. Circ Res 103:343–351PubMedCrossRefGoogle Scholar
  34. 34.
    Levine B, Sinha S, Kroemer G (2008) Bcl-1 family members: dual regulators of apoptosis and autophagy. Autophagy 4:600–606PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  1. 1.Department of PhysiologyUniversity of Pretoria, PretoriaPretoriaSouth Africa

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