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

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.

• Authors: Jikai Yin, Zhongliang Cai, Li Zhang, Jian Zhang, Xianli He, Xilin Du, Qing Wang, Jianguo Lu
• Journal: International journal of oncology (Int J Oncol) Vol. 42 Issue 3 Pg. 1061-9 (Mar 2013) ISSN: 1791-2423 [Electronic] Greece
• PMID: 23314696 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

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

Topics

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