A recombined fusion protein PTD-Grb2-SH2 inhibits the proliferation of breast cancer cells in vitro

Int J Oncol. 2013 Mar;42(3):1061-9. doi: 10.3892/ijo.2013.1768. Epub 2013 Jan 10.

Abstract

The growth factor receptor bound protein 2 (Grb2) is one of the affirmative targets for cancer therapy, especially for breast cancer. In this study, we hypothesized the Src-homology 2 (SH2) domain in Grb2 may serve as a competitive protein-binding agent to interfere with the proliferation of breast cancer cells in vitro. We designed, constructed, expressed and purified a novel fusion protein containing the protein transduction domain (PTD) and Grb2-SH2 domain (we named it after PTD-Grb2-SH2). An immunofluorescence assay was used to investigate the location of PTD-Grb2-SH2 in cells. MTT assay and EdU experiments were applied to detect the proliferation of breast cancer cells. The ultra-structure was observed using transmission electron microscopy. Flow cytometry was used to determine the cytotoxicity of PTD-Grb2-SH2 on cell proliferation. We successfully obtained the PTD-Grb2-SH2 fusion protein in soluble form using a prokaryotic expression system. The new fusion protein successfully passed through both the cellular and nuclear membranes of breast cancer cells. The MTT assay showed that PTD-Grb2-SH2 exhibited significant toxicity to breast cancer cells in a dose- and time-dependent manner in vitro. EdU identified the decreased proliferation rates in treated MDA-MB-231 and SK-BR-3 cells. Observation by transmission electron microscopy and flow cytometry further confirmed the cytotoxicity as apoptosis. Our results show that the HIV1-TAT domain is a useful tool for transporting a low molecular weight protein across the cell membrane in vitro. The PTD-Grb2-SH2 may be a novel agent for breast cancer therapy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis
  • Breast Neoplasms / metabolism*
  • Cell Line, Tumor
  • Cell Proliferation
  • Cell Survival
  • Female
  • GRB2 Adaptor Protein / genetics*
  • GRB2 Adaptor Protein / metabolism
  • Gene Expression
  • Humans
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins* / biosynthesis
  • Recombinant Fusion Proteins* / genetics
  • Recombinant Fusion Proteins* / metabolism
  • Transduction, Genetic
  • src Homology Domains / genetics*

Substances

  • GRB2 Adaptor Protein
  • GRB2 protein, human
  • Recombinant Fusion Proteins