Skip to main content

Advertisement

SpringerLink
Log in

Covalent Immobilization of Human Placental 17β-Hydroxysteroid Dehydrogenase Type 1 onto Glutaraldehyde Activated Silica Coupled with LC-TOF/MS for Anti-Cancer Drug Screening Applications

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), a potential target in breast cancer prevention and therapy, was extracted from human placenta and immobilized on nonporous silica (∼5 μm) with a covalent method for the first time. The optimum initial enzyme concentration and immobilization time during the immobilization process were 0.42 mg mL−1 and 12 h, repectively. The binding was confirmed by scanning electron microscope (SEM) and infrared spectroscopy (FT-IR). It could improve the pH, thermal and storage stability compared to free enzyme. Moreover, the immobilized enzyme could be reused at least four times. A screening method based on it coupled with liquid chromatography–time-of-flight mass spectrometer (LC-TOF/MS) was established, and the half-maximal inhibitory concentration (IC 50) of apigenin for the immobilized enzyme was 291 nM. Subsequently, 10 natural products were evaluated leading to inhibition of the activity of 17β-HSD1 at the concentration of 25 μM, and six of them inhibit the activity over 50%.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12

Similar content being viewed by others

References

  1. Yan, L., Liu, J., Liu, X., Xing, K., Yun, W., Li, F., & Yao, L. (2006). Resveratrol-induced cell inhibition of growth and apoptosis in MCF7 human breast cancer cells are associated with modulation of phosphorylated akt and caspase-9. Applied Biochemistry & Biotechnology, 135, 181–192.

    Article  Google Scholar 

  2. Fournier, D., Poirier, D., Mazumdar, M., & Lin, S. X. (2008). Design and synthesis of bisubstrate inhibitors of type 1 17β-hydroxysteroid dehydrogenase: overview and perspectives. European Journal of Medicinal Chemistry, 43, 2298–2306.

    Article  CAS  Google Scholar 

  3. Aka, J. A., Mazumdar, M., Chen, C. Q., Poirier, D., & Lin, S. X. (2010). 17beta-hydroxysteroid dehydrogenase type 1 stimulates breast cancer by dihydrotestosterone inactivation in addition to estradiol production. Molecular Endocrinology, 24, 832–845.

    Article  CAS  Google Scholar 

  4. Kasai, T., Shozu, M., Murakami, K., Segawa, T., Shinohara, K., Nomura, K., & Inoue, M. (2004). Increased expression of type I 17beta-hydroxysteroid dehydrogenase enhances in situ production of estradiol in uterine leiomyoma. Journal of Clinical Endocrinology & Metabolism, 89, 5661–5668.

    Article  CAS  Google Scholar 

  5. Jarabak, J., Adams, J. A., Williamsashman, H. G., & Talalay, P. (1962). Purification of a 17beta-hydroxysteroid dehydrogenase of human placenta and studies on its transhydrogenase function. Journal of Biological Chemistry, 237, 345–357.

    CAS  Google Scholar 

  6. Adams, J. A., Jarabak, J., & Talalay, P. (1962). The steroid specificity of the 17β-hydroxysteroid dehydrogenase of human placenta. Journal of Biological Chemistry, 237, 3069–3073.

    CAS  Google Scholar 

  7. Langer, L. J., & Engel, L. L. (1958). Human placental estradiol-17 beta dehydrogenase. I. Concentration, characterization and assay. Journal of Biological Chemistry, 233, 583–588.

    CAS  Google Scholar 

  8. Zhu, S. J., Li, Y., Li, H., Wang, Y. L., Xiao, Z. J., Vihko, P., & Piao, Y. S. (2002). Retinoic acids promote the action of aromatase and 17beta-hydroxysteroid dehydrogenase type 1 on the biosynthesis of 17beta-estradiol in placental cells. Journal of Endocrinology, 172, 31–43.

    Article  CAS  Google Scholar 

  9. Breton, R., Housset, D., Mazza, C., & Fontecilla-Camps, J. C. (1996). The structure of a complex of human 17β-hydroxysteroid dehydrogenase with estradiol and NADP + identifies two principal targets for the design of inhibitors. Structure, 4, 905–915.

    Article  CAS  Google Scholar 

  10. Shi, R., & Lin, S. X. (2004). Cofactor hydrogen bonding onto the protein main chain is conserved in the short chain dehydrogenase/reductase family and contributes to nicotinamide orientation. Journal of Biological Chemistry, 279, 16778–16785.

    Article  CAS  Google Scholar 

  11. Puranen, T., Poutanen, M., Ghosh, D., Vihko, R., & Vihko, P. (1997). Origin of substrate specificity of human and rat 17β-hydroxysteroid dehydrogenase type 1, using chimeric enzymes and site-directed substitutions 1. Endocrinology, 138, 3532–3539.

    Article  CAS  Google Scholar 

  12. Lin, S.-X., Shi, R., Qiu, W., Azzi, A., Zhu, D.-W., Al Dabbagh, H., & Zhou, M. (2006). Structural basis of the multispecificity demonstrated by 17β-hydroxysteroid dehydrogenase types 1 and 5. Molecular & Cellular Endocrinology, 248, 38–46.

    Article  CAS  Google Scholar 

  13. Fournier, D., Poirier, D., Mazumdar, M., & Lin, S. X. (2008). Design and synthesis of bisubstrate inhibitors of type 1 17beta-hydroxysteroid dehydrogenase: overview and perspectives. European Journal of Medicinal Chemistry, 43, 2298–2306.

    Article  CAS  Google Scholar 

  14. Bail, J. C. L., Champavier, Y., Chulia, A. J., & Habrioux, G. (2000). Effects of phytoestrogens on aromatase, 3β and 17β-hydroxysteroid dehydrogenase activities and human breast cancer cells. Life Sciences, 66, 1281–1291.

    Article  Google Scholar 

  15. Poirier, D. (2003). Inhibitors of 17β-hydroxysteroid dehydrogenases. Current Medicinal Chemistry, 10, 453–477.

    Article  CAS  Google Scholar 

  16. Michiels, P. J., & Cstephan, L. (2009). Ligand-based NMR spectra demonstrate an additional phytoestrogen binding site for 17beta-hydroxysteroid dehydrogenase type 1. Journal of Steroid Biochemistry & Molecular Biology, 117, 93–98.

    Article  CAS  Google Scholar 

  17. Deluca, D., Krazeisen, A., Breitling, R., Prehn, C., Möller, G., & Adamski, J. (2005). Inhibition of 17beta-hydroxysteroid dehydrogenases by phytoestrogens: comparison with other steroid metabolizing enzymes. Journal of Steroid Biochemistry & Molecular Biology, 93, 285–292.

    Article  CAS  Google Scholar 

  18. Starčević, Š., Turk, S., Brus, B., Cesar, J., Rižner, T. L., & Gobec, S. (2011). Discovery of highly potent, nonsteroidal 17β-hydroxysteroid dehydrogenase type 1 inhibitors by virtual high-throughput screening. The Journal of Steroid Biochemistry and Molecular Biology, 127, 255–261.

    Article  Google Scholar 

  19. Zanin, G. M., & Moraes, F. F. D. (1998). Thermal stability and energy of deactivation of free and immobilized amyloglucosidase in the saccharification of liquefied cassava starch. Applied Biochemistry & Biotechnology, 70-72, 383–394.

    Article  CAS  Google Scholar 

  20. Villee, C. A., & Gordon, E. E. (1955). Further studies on the action of estradiol in vitro. Journal of Biological Chemistry, 216, 203–214.

    CAS  Google Scholar 

  21. Kruger, N. J. (1994) In Basic protein and peptide protocols (pp. 9–15) Springer.

  22. Mohamad, N. R., Marzuki, N. H., Buang, N. A., Huyop, F., & Wahab, R. A. (2015). An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotechnology Biotechnological Equipment, 29, 205–220.

    Article  CAS  Google Scholar 

  23. Petkova, G. A., Záruba, K., & Král, V. (2012). Synthesis of silica particles and their application as supports for alcohol dehydrogenases and cofactor immobilizations: conformational changes that lead to switch in enzyme stereoselectivity. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1824, 792–801.

    Article  CAS  Google Scholar 

  24. Shen, H., Pan, S., Zhang, Y., Huang, X., & Gong, H. (2012). A new insight on the adsorption mechanism of amino-functionalized nano-Fe 3 O 4 magnetic polymers in Cu (II), Cr (VI) co-existing water system. Chemical Engineering Journal, 183, 180–191.

    Article  CAS  Google Scholar 

  25. Chang, Q., & Tang, H. (2014). Immobilization of horseradish peroxidase on NH2-modified magnetic Fe3O4/SiO2 particles and its application in removal of 2, 4-dichlorophenol. Molecules, 19, 15768–15782.

    Article  CAS  Google Scholar 

  26. Klein, M. P., Nunes, M. R., Rodrigues, R. C., Benvenutti, E. V., Costa, T. M., Hertz, P. F., & Ninow, J. L. (2012). Effect of the support size on the properties of β-galactosidase immobilized on chitosan: advantages and disadvantages of macro and nanoparticles. Biomacromolecules, 13, 2456–2464.

    Article  CAS  Google Scholar 

  27. Le Bail, J., Laroche, T., Marre-Fournier, F., & Habrioux, G. (1998). Aromatase and 17β-hydroxysteroid dehydrogenase inhibition by flavonoids. Cancer Letters, 133, 101–106.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors gratefully acknowledge the financial support for this work from the Natural Science Foundation of Jiangsu Province (No. BK20130654) and the National Natural Science Foundation of PR China (No. 81402900).

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China

    Yin Bai, Wen-Di Zhou, Qian Zhang, Chen Yu, Bin Di & Meng-Xiang Su

  2. Key Laboratory on Protein Chemistry and Structural Biology, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China

    Yin Bai, Wen-Di Zhou, Qian Zhang, Chen Yu, Bin Di & Meng-Xiang Su

  3. The Second Affiliated Hospital of Nanjing Medical University, Nanjing, China

    Xian-Min Mu

Authors
  1. Yin Bai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wen-Di Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xian-Min Mu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qian Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chen Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bin Di
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Meng-Xiang Su
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Bin Di or Meng-Xiang Su.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bai, Y., Zhou, WD., Mu, XM. et al. Covalent Immobilization of Human Placental 17β-Hydroxysteroid Dehydrogenase Type 1 onto Glutaraldehyde Activated Silica Coupled with LC-TOF/MS for Anti-Cancer Drug Screening Applications. Appl Biochem Biotechnol 182, 482–494 (2017). https://doi.org/10.1007/s12010-016-2339-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-016-2339-6

Keywords

Search

Navigation