Skip to main content
  • Research Article
  • Published:

The in vitro effects of 2-methoxyestradiol-bis-sulphamate on cell numbers, membrane integrity and cell morphology, and the possible induction of apoptosis and autophagy in a non-tumorigenic breast epithelial cell line

Abstract

2-methoxyestradiol (2ME2) exerts estrogen receptor-independent anti-proliferative, anti-angiogenic and anti-tumor activity in vitro and in vivo. Due to its low bioavailability and rapid metabolic degradation, several analogues have been developed in recent years. 2-methoxyestradiol-bis-sulphamate (2-MeOE2bisMATE) is a bis-sulphamoylated derivative of 2ME2 with anti-proliferative activity. The aim of this study was to investigate cell signaling events induced by 2-MeOE2bisMATE in a non-tumorigenic cell line (MCF-12A) by analysing its influence on cell number, morphology and membrane integrity, and the possible induction of apoptosis and autophagy. Dose- and time-dependent studies revealed that 48 h exposure to 2-MeOE2bisMATE (0.4 μM) resulted in a decrease in cell numbers to 79%. A slight increase in the level of lactate dehydrogenase production was observed in the 2-MeOE2bisMATE-treated cells. Morphological studies revealed an increase in the number of cells in metaphase. Hallmarks of apoptosis were also found, namely nuclear fragmentation and apoptotic bodies. In addition, increased lysosomal staining was observed via fluorescent microscopy, suggesting the induction of another type of cell death, namely autophagy. Since 2-MeOE2bisMATE is regarded as a potential anti-cancer agent, it is also imperative to investigate the susceptibility of non-tumorigenic cells to its influence. The data generated from this study contributes to the understanding of the action that 2-MeOE2bisMATE exerts on the non-tumorigenic MCF-12A breast epithelial cell line.

Abbreviations

2ME2:

2-methoxyestradiol

2-MeOE2bisMATE:

2-methoxyestradiol-bis-sulphamate

adiol:

androstenediol

CA II:

carbonic anhydrase II

DHEA:

dehydroepiandrostene

DMSO:

dimethyl sulphoxide

LDH:

lactate dehydrogenase

PBS:

phosphate buffered saline

PlasDIC:

optical transmitted light differential interference contrast

STS:

steroid sulphatase

TEM:

transmission electron microscopy

References

  1. Dubey, R.K. and Jackson, E.K. Potential vascular actions of 2-methoxyestradiol. Trends Endocrinol. Metab. 20 (2009) 374–379.

    Article  CAS  PubMed  Google Scholar 

  2. Pribluda, V.S., Gubish, E.R., La Vallee, T.M., Treston, A., Swartz, G.M and Green, S.J. 2-Methylestradiol: An endogenous antiangiogenic and antiproliferative drug candidate. Cancer Metastasis Rev. 19 (2000) 173–179.

    Article  CAS  PubMed  Google Scholar 

  3. Bhati, R., Gokmen-Polar, Y., Sledge, G.W., Fan, C., Nakshatri, H., Ketelsen, D., Borchers, C.H., Dial, M.J., Patterson, C. and Klauber-DeMore, N. 2-Methoxyestradiol inhibits the anaphase-promoting complex and protein translation in human breast cancer cells. Cancer Res. 67 (2007) 702–708.

    Article  CAS  PubMed  Google Scholar 

  4. Banerjee, S.N., Sengupta, K., Banerjee, S., Saxene, N.K. and Banerjee, S.K. 2-Methoxyestradiol exhibits a biphasic effect on VEGF-A in tumor cells and upregulation is mediated through ER-α: a possible signaling pathway associated with the impact of 2-ME2 on proliferating cells. Neoplasia 5 (2003) 417–426.

    CAS  PubMed  Google Scholar 

  5. Funakoshi, T., Birsner, A.E. and D’Amato, R.J. Antiangiogenic effect of oral 2-methoxyestradiol on choroidal neovascularization in mice. Exp. Eye Res. 83 (2006) 1102–1107.

    Article  CAS  PubMed  Google Scholar 

  6. Matei, D., Schilder, J., Sutton, G., Perkins, S., Breen, T., Quon, C. and Sidor, C. Activity of 2 methoxyestradiol (Panzem® NCD) in advanced, platinum-resistant ovarian cancer and primary peritoneal carcinomatosis: A hoosier oncology group trial. Gynaecol. Oncol. 115 (2009) 90–96.

    Article  CAS  Google Scholar 

  7. Lakka, S.S. and Rao, J.S. Angiogenic therapy in brain tumors. Expert Rev. Neurother. 8 (2008) 1457–1473.

    Article  PubMed  Google Scholar 

  8. Sutherland, T.E., Anderson, R.L., Hughes, R.A., Altmann, E., Schuliga, M., Ziogas, J. and Stewart, A.G. 2-Methoxyestradiol- a unique blend of activities generating a new class of anti-tumour/anti-inflammatory agents. Drug Discov. Today 12 (2007) 577–584.

    Article  CAS  PubMed  Google Scholar 

  9. Thaver, V., Lottering, M.L., Van Papendorp, D. and Joubert, A. In vitro effects of 2-methoxyestradiol on cell numbers, morphology, cell cycle progression, and apoptosis induction in oesophageal carcinoma cells. Cell Biochem. Funct. 27 (2009) 205–210.

    Article  CAS  PubMed  Google Scholar 

  10. Lakhani, N.J., Lepper, E.R., Sparreboom, A., Dahut, W.L., Venitz, J. and Figg, W.D. Determination of 2-methxyestradiol in human plasma, using liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass. Spectrom. 19 (2005) 1176–1182.

    Article  CAS  PubMed  Google Scholar 

  11. Foster, P.A., Ho, Y.T., Newman, S.P., Kasprzyk, M.P., Leese, M.P., Potter, B.V., Reed, M.J. and Purohit, A. 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 (2008) 251–260.

    Article  CAS  PubMed  Google Scholar 

  12. Reed, M.J., Purohit, A., Woo, L.W.L., Newman, S.P. and Potter, B.V.L. Steroid sulfatase: molecular biology, regulation, and inhibition. Endocr. Rev. 26 (2005) 171–202.

    Article  CAS  PubMed  Google Scholar 

  13. Kirches, E. and Wraich-Kirches, M. 2-Methoxyestradiol as a potential cytostatic drug in gliomas? Anticancer Agents Med. Chem. 9 (2009) 55–65.

    CAS  PubMed  Google Scholar 

  14. Utsumi, T., Leese, M.P., Chander, S.K., Gaukroger, K., Purohit, A., Newman, S.P., Potter B.V. and Reed, M.J. The effects of 2-methoxyoestrogen sulphamates on the in vitro and in vivo proliferation of breast cancer cells. J. Steroid Biochem. Mol. Biol. 94 (2005) 219–227.

    Article  CAS  PubMed  Google Scholar 

  15. Tagg, S.L., Foster, P.A., Leese, M.P., Potter, B.V., Reed, M.J., Purohit, A. and Newman, S.P. 2-methoxyoestradiol-3,27-O,O-bis-sulphamate and 2-deoxy-D-glucose in combination: a potential treatment for breast and prostate cancer. Br. J. Cancer 99 (2008) 1842–1848.

    Article  CAS  PubMed  Google Scholar 

  16. Ireson, C.R., Chander, S.K., Purohit, A., Perera, S., Newman, S.P., Parish, D., Leese, M.P., Smith, A.C., Potter, B.V. and Reed, M.J. Pharmacokinetics and efficacy of 2-methoxyoestradiol and 2-methoxyoestradiol-bis-sulphamate in vivo in rodents. Br. J. Cancer 90 (2004) 932–937.

    Article  CAS  PubMed  Google Scholar 

  17. Suzuki, R.N., Newman, S.P., Purohit, A., Leese, M.P., Potter, B.V. and Reed, M.J. 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 (2003) 269–278.

    Article  CAS  PubMed  Google Scholar 

  18. Foster, P.A., Newman, S.P., Leese, M.P., Bernetiere, S., Diolez, C., Camara, J., Hacher, B., Baronnet, M.M., Ali, T., Potter, B.V., Reed, M.J. and Purohit, A. A new micronized formulation of 2-methoxyestradiol-bis-sulphamate (STX140) is therapeutically potent against breast cancer. Anticancer Res. 28 (2008) 577–581.

    CAS  PubMed  Google Scholar 

  19. Wood, L., Leese, M.P., Mouzakiti, A., Purohit, A., Potter, B.V., Reed, M.J. and Packham, G. 2-MeOE2bisMATE induces caspase-dependent apoptosis in CAL51 breast cancer cells and overcomes resistance to TRAIL via cooperative activation of caspases. Apoptosis 9 (2004) 323–332.

    Article  CAS  PubMed  Google Scholar 

  20. Newman, S.P., Leese, M.P., Purohit, A., James, D.R.C., Rennie, C.E., Potter, B.V. and Reed, M.J. Inhibition of in vitro angiogenesis by 2-Methoxy- and 2-ethyl-estrogen sulphamates. Int. J. Cancer 109 (2004) 533–540.

    Article  CAS  PubMed  Google Scholar 

  21. Joubert, A.M., Marais, S. and Maritz, C. Influence of 2-methoxyestradiol on MCF-7 cells: An improved differential interference contrasting technique and Bcl-2 and Bax protein expression levels. Biocell 33 (2009) 67–70.

    CAS  PubMed  Google Scholar 

  22. Gillies, R.J., Didier, N. and Denton, M. Determination of cell number in monolayer cultures. Anal. Biochem. 159 (1986) 109–113.

    Article  CAS  PubMed  Google Scholar 

  23. Grever, M.R., Schepartz, S.A. and Chabner, B.A. The National Cancer Institute: cancer drug discovery and development program. Semin. Oncol. 19 (1992) 622–638.

    CAS  PubMed  Google Scholar 

  24. Kusuzaki, K., Murata, H., Takeshita, H., Hashiguchi, S., Nozaki, T., Emoto, K., Ashihara, T. and Hirasawa, Y. Intracellular binding sites of acridine orange in living osteosarcoma cells. Anticancer Res. 20 (2000) 971–975.

    CAS  PubMed  Google Scholar 

  25. Elmore, S. Apoptosis: a Review of programmed cell death. Toxicol. Pathol. 35 (2007) 495–516.

    Article  CAS  PubMed  Google Scholar 

  26. Xia, GH., Chen, B.A., Shao, Z.Y., Lu, H.X., Konstanze, D. and Hartmut, D. Mechanism of 2-methoxyestradiol-induced apoptosis in myelodysplastic syndrome MUTZ-1 cell line. Zhongguo Shi Yan Xue Ye Xue Za Zhi 15 (2007) 296–301.

    CAS  PubMed  Google Scholar 

  27. Kachadourian, R., Liochev, S.I., Cabelli, D.E., Patel, M.N., Fridovich, I. and Day, B.J. 2-Methoxyestradiol does not inhibit superoxide dismutase. Arch. Biochem. Biophys. 392 (2001) 349–353.

    Article  CAS  PubMed  Google Scholar 

  28. Fu, J., Weise, A.M., Falany, J.L., Falany, C.N., Thibodeau, B.J., Miller, F.R., Korarek, T.A. and Runge-Morris, M. Expression of estrogenicity genes in a lineage cell culture model of human breast cancer progression. Breast Cancer Res. Treat. 120 (2010) 35–40.

    Article  CAS  PubMed  Google Scholar 

  29. Yoshimura, N., Harada, N., Bukholm, I., Karesen, R., Borresen-Dale, A. and Kristensen, V.N. Intratumoural mRNA expression of genes from the oestradiol metabolic pathway and clinical and histopathological parameters of breast cancer. Breast Cancer Res. 6 (2004) R46–R55.

    Article  CAS  PubMed  Google Scholar 

  30. Stanway, S.J., Delavault, P., Purohit, A., Woo, L.W., Thurieau, C., Potter, B.V. and Reed, M.J. Steroid sulfatase: a new target for the endocrine therapy of breast cancer. Oncologist 12 (2007) 370–374.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Annie M. Joubert.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Visagie, M.H., Joubert, A.M. The in vitro effects of 2-methoxyestradiol-bis-sulphamate on cell numbers, membrane integrity and cell morphology, and the possible induction of apoptosis and autophagy in a non-tumorigenic breast epithelial cell line. Cell Mol Biol Lett 15, 564–581 (2010). https://doi.org/10.2478/s11658-010-0030-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-010-0030-4

Key words