Deciphering therapeutic potential of PEGylated recombinant PTEN-silver nanoclusters ensemble on 3D spheroids


The therapeutic application of recombinant proteins is limited due to their inherent structural complexity. Additionally, screening of therapeutic potential of protein products requires an appropriate testing platform to achieve biological relevance. Fabrication of three dimensional cultures bridges the gap between in vitro based monolayer cultures and clinical applications. In this perspective, glioblastoma U-87 MG and breast cancer MCF7 spheroids were generated to assess the therapeutic prospect of recombinant PTEN protein. PTEN bound to silver nanoclusters was encapsulated within PEG coating, which resulted in fabrication of spherical nanocarriers named as PTEN-nanocomposites. Internalization of PTEN-nanocomposites in the spheroids was confirmed by confocal microscopy. Upon uptake, PTEN-nanocomposites led to modulation of cyclins and apoptosis gene regulators culminating in cell cycle arrest and reduced cell viability as confirmed by calcein-AM/PI dual staining and alamar blue assay. Further, combination of tamoxifen and PTEN-nanocomposites on U-87 MG spheroids resulted in two-fold reduction of drug dosage. The study revealed that the monolayer culture results translated to the 3D culture as well, however higher dose of the recombinant PTEN was required for the spheroid system. The anti-proliferative role of PTEN-nanocomposites in a complex 3D environment augments its biological implication and paves the way for recombinant PTEN based therapeutic applications.

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  1. 1.

    Fennema E, Rivron N, Rouwkema J, van Blitterswijk C, de Boer J (2013) Spheroid culture as a tool for creating 3D complex tissues. Trends Biotechnol 31:108–115

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Pampaloni F, Reynaud EG, Stelzer EHK (2007) The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol 8:839

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Cunha C, Panseri S, Villa O, Silva D, Gelain F (2011) 3D culture of adult mouse neural stem cells within functionalized self-assembling peptide scaffolds. Int J Nanomed 6:943–955

    CAS  Article  Google Scholar 

  4. 4.

    Owen SC, Shoichet MS (2010) Design of three-dimensional biomimetic scaffolds. J Biomed Mater Res, Part A 94:1321–1331

    Google Scholar 

  5. 5.

    Breslin S, O’Driscoll L (2013) Three-dimensional cell culture: the missing link in drug discovery. Drug Discov Today 18:240–249

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Amaral RLF, Miranda M, Marcato PD, Swiech K (2017) Comparative analysis of 3D bladder tumor spheroids obtained by forced floating and hanging drop methods for drug screening. Front Physiol 8:605

    Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Frey O, Misun PM, Fluri DA, Hengstler JG, Hierlemann A (2014) Reconfigurable microfluidic hanging drop network for multi-tissue interaction and analysis. Nat Commun 5:4250

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Zanoni M, Piccinini F, Arienti C, Zamagni A, Santi S, Polico R et al (2016) 3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Sci Rep 6:19103

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Riedl A, Schlederer M, Pudelko K, Stadler M, Walter S, Unterleuthner D et al (2017) Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT–mTOR–S6 K signaling and drug responses. J Cell Sci 130:203

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Song MS, Salmena L, Pandolfi PP (2012) The functions and regulation of the PTEN tumour suppressor. Nat Rev Mol Cell Biol 13:283–296

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Hollander MC, Blumenthal GM, Dennis PA (2011) PTEN loss in the continuum of common cancers, rare syndromes and mouse models. Nat Rev Cancer 11:289–301

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Stambolic V, Suzuki A, de la Pompa JL, Brothers GM, Mirtsos C, Sasaki T et al (1998) Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 95:29–39

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Papa A, Chen M, Pandolfi PP (2013) Pills of PTEN? In and out for tumor suppression. Cell Res 23:1155–1156

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Arora N, Gavya SL, Ghosh SS (2018) Multi-facet implications of PEGylated lysozyme stabilized-silver nanoclusters loaded recombinant PTEN cargo in cancer theranostics. Biotechnol Bioeng 115:1116–1127

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Arora N, Ghosh SS (2016) Functional characterizations of interactive recombinant PTEN-silica nanoparticles for potential biomedical applications. RSC Adv 6:114944–114954

    CAS  Article  Google Scholar 

  16. 16.

    Zhou T, Huang Y, Li W, Cai Z, Luo F, Yang CJ et al (2012) Facile synthesis of red-emitting lysozyme-stabilized Ag nanoclusters. Nanoscale 4:5312–5315

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Ivascu A, Kubbies M (2006) Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis. J Biomol Screen 11:922–932

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Friedrich J, Seidel C, Ebner R, Kunz-Schughart LA (2009) Spheroid-based drug screen: considerations and practical approach. Nat Protoc 4:309–324

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Walzl A, Unger C, Kramer N, Unterleuthner D, Scherzer M, Hengstschlager M et al (2014) The resazurin reduction assay can distinguish cytotoxic from cytostatic compounds in spheroid screening assays. J Biomol Screen 19:1047–1059

    Article  PubMed  Google Scholar 

  20. 20.

    Riccardi C, Nicoletti I (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 1:1458–1461

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Froehlich K, Haeger JD, Heger J, Pastuschek J, Photini SM, Yan Y et al (2016) Generation of multicellular breast cancer tumor spheroids: comparison of different protocols. J Mammary Gland Biol Neoplasia 21:89–98

    Article  PubMed  Google Scholar 

  22. 22.

    Collazo A, Bricaud O, Desai K (2005) Use of confocal microscopy in comparative studies of vertebrate morphology. Methods Enzymol 395:521–543

    Article  PubMed  Google Scholar 

  23. 23.

    Chan LL, Wilkinson AR, Paradis BD, Lai N (2012) Rapid image-based cytometry for comparison of fluorescent viability staining methods. J Fluoresc 22:1301–1311

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Maddika S, Ande SR, Wiechec E, Hansen LL, Wesselborg S, Los M (2008) Akt-mediated phosphorylation of CDK2 regulates its dual role in cell cycle progression and apoptosis. J Cell Sci 121:979–988

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Hiraoka D, Aono R, Hanada S-I, Okumura E, Kishimoto T (2016) Two new competing pathways establish the threshold for cyclin-B-Cdk1 activation at the meiotic G2/M transition. J Cell Sci 129:3153–3166

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Katsuno Y, Suzuki A, Sugimura K, Okumura K, Zineldeen DH, Shimada M et al (2009) Cyclin A-Cdk1 regulates the origin firing program in mammalian cells. Proc Natl Acad Sci 106:3184

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Bendris N, Lemmers B, Blanchard J-M, Arsic N (2011) Cyclin A2 mutagenesis analysis: a new insight into CDK activation and cellular localization requirements. PLoS ONE 6:e22879

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Westphal D, Dewson G, Czabotar PE, Kluck RM (2011) Molecular biology of Bax and Bak activation and action. Biochem Biophys Acta 1813:521–531

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Tsuruta F, Masuyama N, Gotoh Y (2002) The phosphatidylinositol 3-kinase (PI3 K)-Akt Pathway suppresses Bax translocation to mitochondria. J Biol Chem 277:14040–14047

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Dansen TB, Burgering BMT (2008) Unravelling the tumor-suppressive functions of FOXO proteins. Trends Cell Biol 18:421–429

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Fu Z, Tindall DJ (2008) FOXOs, cancer and regulation of apoptosis. Oncogene 27:2312

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Zhang X, Tang N, Hadden TJ, Rishi AK (2011) Akt, FoxO and regulation of apoptosis. Biochem Biophys Acta 1813:1978–1986

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Medema RH, Kops GJPL, Bos JL, Burgering BMT (2000) AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 404:782

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Huang Y, Sramkoski RM, Jacobberger JW (2013) The kinetics of G2 and M transitions regulated by B cyclins. PLoS ONE 8:e80861

    Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Krishan A (1975) Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 66:188

    CAS  Article  PubMed  Google Scholar 

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Financial supports of the Department of Biotechnology, Government of India, for the NER-BPMC (BT/PR 25095/NER/95/1011/2017), DBT Program Support (BT/PR13560/COE/34/44/2015), partial support of the Department of Electronics and Information Technology (No. 5(9)/2012-NANO (Vol. II)), Government of India are acknowledged. Authors also acknowledge the support of the Central Instruments Facility (CIF) and the Centre for Nanotechnology, IIT Guwahati for providing instrumentation facilities.

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Correspondence to Siddhartha Sankar Ghosh.

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Arora, N., Shome, R. & Ghosh, S.S. Deciphering therapeutic potential of PEGylated recombinant PTEN-silver nanoclusters ensemble on 3D spheroids. Mol Biol Rep 46, 5103–5112 (2019).

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  • Anti-proliferative activity
  • Combination therapy
  • PEG coating
  • Recombinant PTEN
  • Spheroid culture
  • Silver nanoclusters